REGULATION OF Na+/H+ EXCHANGER (NHE1) STABILITY IN PTEN-/- MOUSE EMBRYONIC FIBROBLASTS JOANNE JAMES B.Sc. Hons (University of Queensland) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. JOANNE JAMES 23 September 2013 i ACKNOWLEDGEMENTS Firstly, I would like to express my sincere appreciation and gratitude towards my supervisor, Associate Professor Marie-Veronique Clement for her constant guidance, patience and support she has given me throughout my graduate study in NUS. Also, I would like to thank my TAC members, Prof Sit Kim Ping and Dr Chen Ee Sin for their invaluable input towards my project. I am extremely grateful to Dr Jaspal, for her guidance and support in my project. In addition, I would like to express my sincere appreciation to Dr Jeya for the constant encouragement and San Min, Giree and the rest of the lab members who have been a great source of support during tough times. Importantly, I would like to thank my fiancé Nathaniel who has stood by me in times of distress and been a pillar of strength for me. Thank you for your patience, love and support always and for bringing out the best in me. Last but never least, I want to express my heartfelt appreciation to my Dad, Mum, brother and sister-in law for their words of encouragement in tough times. I am extremely grateful to my wonderful parents for their unconditional love, support and for believing in me. Thank you for teaching me to be resilient towards achieving my dreams. Finally, I dedicate this dissertation to my parents. ii TABLE OF CONTENTS DECLARATION . i ACKNOWLEDGEMENTS . ii TABLE OF CONTENTS . iii SUMMARY . vii LIST OF TABLES ix LIST OF FIGURES ix LIST OF ABBREVIATIONS xii CHAPTER 1: INTRODUCTION . 1.1 Na+/H+ exchanger-1 (NHE1) . 1.1.1 NHE1 isoforms and NHE1 structure . 1.1.2 Regulation of NHE1 . 1.1.3. Physiological function of NHE1 . 1.1.4 Role of NHE1 in cancer progression 11 1.1.5 Link between NHE1 and PI3K/Akt pathway in cell survival 14 1.2 PI3-Kinase (PI3K)/Akt Pathway 16 1.2.1 Structure of Akt and its isoforms 16 1.2.2 Activation of Akt . 18 1.2.3 Negative regulation of PI3K/Akt pathway . 21 1.2.4 Physiological role of Akt pathway . 23 1.2.5 Akt in tumourigenesis . 26 AIM OF STUDY . 32 CHAPTER 2: MATERIALS AND METHODS . 33 2.1 Materials . 33 2.1.1 Chemicals and reagents 33 2.1.2 Antibodies 34 iii 2.1.3 Cell lines and cell culture . 35 2.2 Methods 35 2.2.1 RNA Interference (RNAi) Assay . 35 2.2.2 RNA Extraction and Real-Time PCR 36 2.2.3 Sodium Dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and Western blot analysis . 38 2.2.4 Membrane/Cytosol fractionation . 39 2.2.5 Immunoprecipitation assay . 40 2.2.6 Statistical Analysis . 41 CHAPTER 3: RESULTS 42 3.1 NHE1 is a Hsp90 client protein . 42 3.1.1 Increase in NHE1 protein expression in MEFPTEN-/- cells compared to MEFPTEN+/+ cells does not correlate with an increase in NHE1 mRNA production . 42 3.1.2 NHE1 protein stability is prolonged in MEFPTEN-/- cells as compared to MEFPTEN+/+ cells . 45 3.1.3 Proteasome inhibition increases NHE1 stability in MEFPTEN+/+ cells but have no effect in MEFPTEN-/- cells . 49 3.1.4 Hsp90 may be involved in stabilizing NHE1 protein expression in MEFPTEN-/- cells 52 3.1.5 Increased binding of Hsp90 and NHE1 protein in MEFPTEN-/- cells . 55 3.1.6 Effect of inhibition of Hsp90 activity on NHE1 expression in MEFPTEN-/- cells 56 3.1.7 Inhibition of Hsp90 destabilizes NHE1 Protein in MEFPTEN-/- cells 65 3.1.8 Hsp90 prevents ubiquitination of NHE1 protein in MEFPTEN-/- cells 67 3.2 Akt is responsible for bringing Hsp90 to the membrane and increasing NHE1 stability in MEFPTEN-/- cells 70 3.2.1 Hyperphosphorylation of Akt correlates with an increased level of NHE1 in MEFPTEN-/- cells . 70 3.2.2 NHE1 binds to Akt in MEFPTEN-/- cells . 87 iv 3.2.3 Akt is a client protein of Hsp90 . 89 3.2.4 NHE1 forms a complex with Hsp90 and Akt in MEFPTEN-/- cells . 102 3.2.5 Hsp90-Akt downregulation affects cell survival in MEFPTEN-/- cells 111 3.3 The NHE1-Hsp90-Akt complex is a model of breast cancer progression . 113 3.3.1 Regulation of NHE1 protein expression in MCF10AT1Kcl.2 series of breast cancer cell lines 113 3.3.2 Stabilisation of Akt induces formation of Hsp90/Akt/NHE1 complex in MCF10AT1 124 3.3.3 Akt regulates NHE1 protein expression in MCF10CA1a 131 3.3.4 NHE1 interacts with Akt in MCF10CA1a and not MCF10CA1h 134 3.3.5 Akt-Hsp90 complex is detectable in MCF10CA1a but is absent in MCF10CA1h breast cancer cells . 136 3.3.6 Hsp90 stabilizes NHE1 protein expression through direct interaction in MCF10Ca1a breast cancer cell lines . 138 3.4 NHE1 regulates Akt phosphorylation at Ser 473 site in MEFPTEN-/- and MCF10CA1a cells 144 3.4.1 Downregulation of NHE1 dephosphorylates Akt at Ser 473 in MEFPTEN-/- and MCF10CA1a cells . 144 CHAPTER 4: DISCUSSION 151 4.1 NHE1 is a potential client protein of Hsp90 in MEFPTEN-/- cells . 151 4.1.1 Increased stability of NHE1 protein in MEFPTEN-/- cells in comparison to MEFPTEN+/+ cells 151 4.1.2 Hsp90 confers protection to NHE1 in MEFPTEN-/- cells 155 4.2 Akt mediates regulation of NHE1 in MEFPTEN-/- cells . 159 4.2.1 Regulation of NHE1 by Akt occurs at a protein level and not transcriptionally in MEFPTEN-/- cells 159 4.2.2 Regulation of NHE1 by Akt in MEFPTEN-/- cells is independent of NHE1 activity 162 4.2.3 NHE1 acts as a scaffold for Akt 163 v 4.2.4 Hsp90 protects hyperphosphorylated Akt from degradation in MEFPTEN-/- cells 164 4.2.5 Hsp90 recruitment to the membrane is dependent on Akt in MEFPTEN-/- cells 165 4.2.6 Hsp90-Akt complex stabilizes NHE1 at the plasma membrane in MEFPTEN-/- cells 166 4.3 Physiological relevance of novel NHE1-Akt-Hsp90 complex in MCF10AT1kcl.2 breast cancer series 169 4.3.1 Detection of novel Hsp90-Akt-NHE1 complex in malignant MCF10CA1a breast epithelial cells . 169 4.3.2 The stability of Akt may play a role in the transition between premalignant MCF10AT1 to malignant MCF10CA1a breast epithelial cells 172 4.4. Regulation of Akt Ser 473 phosphorylation by NHE1 in both MEFPTEN-/cells and MCF10CA1a breast epithelial cells . 174 4.4.1 NHE1 scaffolding function promotes the regulation of Akt Ser 473 phosphorylation in both MEFPTEN-/- cells and MCF10CA1a breast epithelial cells . 174 CONCLUSION . 177 REFERENCES 180 APPENDICES . 197 PUBLICATION AND PRESENTATIONS . 199 vi SUMMARY The Na+/H+ Exchanger (NHE1), a membrane antiporter, has been implicated in tumour cell survival. However, the regulation of NHE1 expression level in tumour cells is not completely understood. Interestingly, the upregulation of NHE1 protein expression in PTEN-null Mouse embryonic fibroblasts (MEFPTEN-/-) compared to PTEN-wildtype Mouse embryonic fibroblasts (MEFPTEN+/+) does not correlate with an increase in NHE1 transcription. Hence, we hypothesized that increased NHE1 expression may be due to increased stability of the protein. In the current study, we show that Heat shock protein 90 (Hsp90) confers stability to NHE1 though enhanced interaction in MEFPTEN-/- cells as compared to MEFPTEN+/+ cells. The inhibition of Hsp90 activity using Geldanamycin and silencing of Hsp90 gene induced a decrease in NHE1 protein level in MEFPTEN-/- cells while no effect was observed in control cells. In addition, NHE1 was found to interact with Akt in MEFPTEN-/- cells. Both inhibition of Akt activation and silencing Akt gene expression decreased NHE1 protein expression. Moreover, our data reveals that Akt is involved in trafficking Hsp90 from the cytosol to the plasma membrane in MEFPTEN-/- cells. Similar result was observed in MCF10CA1a, a breast carcinoma cancer cell line. Additionally, we found that NHE1 may be involved in the regulation of Akt Serine 473 phosphorylation. We show that this occurs through the complex formation between NHE1, mTOR, Rictor in both MEFPTEN-/- and MCF10CA1a vii cells. Taken together, this study establishes a possible tripartite complex between NHE1, Akt and Hsp90 that may be critical for stabilizing NHE1 expression level and its role in survival of tumour cells. We propose that our findings might be critical for the development and maintenance of tumour cells. Indeed, results from this study could establish a new strategy to consider when developing new chemotherapeutic or even chemo-preventive drugs. viii LIST OF TABLES Table 1. Summary of studies indicating tissue distribution and intracellular localization of NHE isoforms. . Table 2. Human NHE1 binding partners and phosphorylation sites LIST OF FIGURES Figure A: Topological model of the Na+/H+ exchanger 1(NHE1). . Figure B. The regulation of NHE1 and its roles in driving tumor hallmark behaviors. 13 Figure C. Schematic diagram of Akt structure. 17 Figure D: Activation and regulation of Akt 20 Figure E: Akt signalling pathway and its role in various biological processes. 25 Figure 1: Increased NHE1 protein expression in MEFPTEN-/- cells as compared to MEFPTEN+/+ cells. 43 Figure 2: Increase in NHE1 protein expression in MEF PTEN-/- cells is not transcriptionally mediated 44 Figure 3: NHE-1 protein is more stable in MEFPTEN-/- cells as compared to MEFPTEN+/+ cells . 48 Figure 4: MG132-mediated inhibition of proteasome degradation complex displays no effect on NHE1 protein expression in MEFPTEN-/- cells 51 Figure 5: Hsp90 is present at the membrane in MEFPTEN-/- cells 54 Figure 6: Enhanced binding of NHE1 to Hsp90 in MEFPTEN-/- cells as compared to MEFPTEN-/- cells . 56 Figure 7: Geldanamycin-induced inhibition of Heat shock protein 90 causes downregulation of NHE1 in MEFPTEN -/- cells 58 Figure 8: Silencing Hsp90 causes downregulation of NHE1 protein expression in both MEFPTEN+/+ and MEFPTEN-/- cells 61 Figure 9: Geldanamycin causes proteasome-mediated degradation of NHE1 in MEFPTEN+/+ and MEFPTEN-/- cells 64 Figure 10: Silencing Hsp90 reduces stability of NHE1 protein in . 66 MEFPTEN-/-cells . 66 Figure 11: Hsp90 inhibition results in ubiquitination of NHE1 69 Figure 12: Hyperphosphorylation of Akt correlates with increased NHE1 protein expression in MEFPTEN-/- cells compared to MEFPTEN+/+ cells. 72 Figure 13: Akt isoform is detectable in MEFPTEN-/- cells 73 Figure 14: Silencing Akt induces downregulation of NHE1 protein expression in MEFPTEN-/- cells. . 75 Figure 15: Silencing Akt shows no effect on NHE1 transcription in MEFPTEN-/cells . 76 Figure 16: LY294002 induces downregulation of NHE1 protein expression in MEFPTEN-/- cells 78 Figure 17: Wortmannin induces downregulation of NHE1 protein expression in MEFPTEN-/- cells 80 Figure 18: API-1 induces downregulation of NHE1 protein expression in MEFPTEN-/- cells 83 ix 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 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(Adapted from Luo 2011, Dissertation) 197 Appendix B. Effect of silencing NHE1 leads to Akt dephosphorylation. MEFPTEN-/- cells (KO) were transfected with NHE1 siRNA (siNHE1) or control siRNA (sico). Twenty four hours post transfection, cells were serum-deprived in DMEM/0.5%FBS (KO 0.5%) for another 24 hrs or grown in DMEM/10%FBS (KO 10%). 48 hours post transfection, cell lysates were subjected to immunoblot blot analysis. A representative blot from three independent experiments is shown. (Adapted from Luo 2011, Dissertation) 198 PUBLICATION AND PRESENTATIONS Publication Joanne James, Jaspal Kaur Kumar and Marie Veronique Clement. Na+/H+ exchanger (NHE1) is a client protein of Hsp90. (In preparation) Joanne James, Jaspal Kaur Kumar and Marie Veronique Clement. Regulation of Akt/NHE1 feedback loop in PTEN-null Mouse Embryonic Fibroblasts. (In preparation) Poster presentations Joanne James, Jaspal Kaur Kumar and Marie Veronique Clement. Regulation of Na+/H+ exchanger (NHE1) expression by hyperactivation of Akt in PTEN-null mouse embryonic fibroblasts. Poster presented at the 102nd Annual Meeting of American Association of Cancer Research (AACR), held at Orlando, Florida, USA (2011). Joanne James, Jaspal Kaur Kumar and Marie Veronique Clement Regulation of Na+/H+ exchanger (NHE1) protein stability in PTEN-null mouse embryonic fibroblasts. Poster presented at the The Cell Symposia: Hallmarks of Cancer, held at San Francisco, California, USA (2012) 199 [...]... detectable in MCF10AT1 and MCF10A1 control cells 12 7 Figure 38: MG132-mediated inhibition induces NHE1-Hsp90-Akt complex in MCF10AT1 cell line 13 0 Figure 39: Effect of LY294002 on NHE1 protein expression in MCF10CA1a 13 2 Figure 40: Akt 1 and 2 are detectable in MCF10CA1a 13 3 Figure 41: Silencing Akt induces downregulation of NHE1 protein expression in MCF10CA1a ... MCF10AT1Kcl.2 series of breast epithelial cells 11 5 Figure 33: Profiling of NHE1 expression and Akt phosphorylation in MCF10AT1Kcl.2 series of breast cancer cell lines 11 7 Figure 34: NHE1 protein stability studies in cells in MCF10A1, MCF10AT1, MCF10CA1a and MCF10CA1h 12 3 Figure 35: Comparison of Akt phosphorylation levels in MCF10A series of breast cancer cell lines 12 5 Figure 36:... Topological model of the Na+/ H+ exchanger 1( NHE1) The diagram depicts the orientation of transmembrane segments I–XII and the N-terminal (N) and C-terminal cytosolic tail (C) of NHE1 Adapted from Slepkov et al 200 519 1. 1.2 Regulation of NHE1 1. 1.2.A Transcriptional regulation of NHE1 To date, a number of transcription factors have been proposed to be involved in the regulation of the NHE1 gene which is... membrane in MCF10Ca1a cells 14 8 Figure 51: NHE1 interacts with mTOR/Rictor complex at the membrane in MEFPTEN-/- cells and MCF10CA1a cells 15 0 19 7 Appendix A: NHE1 expression but not activity is involved in regulation of Akt Phosphorylation 19 7 Appendix B Effect of silencing NHE1 leads to Akt dephosphorylation 19 8 xi LIST OF ABBREVIATIONS 10 A1 MCF10A1 non-tumourigenic... MEFPTEN+/+ PTEN- wildtype Mouse Embryonic Fibroblasts MEF Mouse embryonic fibroblast mTOR Mammalian target of rapamycin mTORC2 Mammalian target of rapamycin Complex 2 NHE Na+/ H+ exchanger NHE1 Na+/ H+ exchanger 1 O2.- Superoxide PDK1 3-phosphoinositide-dependent protein kinase -1 xii PHLPP PH domain leucine-rich repeat protein phosphatase PIP2 Phosphatidylinositol-3,4-bisphosphate PIP3 Phosphatidylinositol-3,4,5-trisphosphate... affect NHE1/Hsp90 complex significantly 10 5 Figure 29: Akt recruits Hsp90 to the membrane in MEFPTEN-/- cells 10 7 Figure 30: Silencing Hsp90 reduces half-life of NHE1 protein in MEFPTEN-/-cells 11 0 Figure 31: Geldanamycin-induced inhibition of Heat shock protein 90 causes increased cell death in MEFPTEN-/- cells as compared to MEFPTEN+/+ cells 11 2 Figure 32: Isolation of MCF10AT1Kcl.2... adhesion (reviewed in 51 ) Intriguingly, the cytoplasmic tail of NHE1 has been shown to act as a scaffold as it contains a vast number of binding sites for interacting partners to bind and elicit many of its functions52 Table 1 entails the list of binding partners of NHE1 and their corresponding binding and phosphorylation sites within the cytoplasmic tail17 8 Abbreviation Interacting Protein ERM AKT ROCK... 13 3 Figure 42: NHE1-Akt interaction is detectable in MCF10CA1a and not in MCF10CA1h cells 13 5 Figure 43: Akt-Hsp90 complex is detected in MCF10CA1a but not MCF10CA1h breast epithelial cells 13 7 Figure 44: Hsp90 is present at the membrane in MCF10CA1a breast cancer cells 13 9 Figure 45: NHE1 interacts with HSP90 in MCF10CA1a cells 14 0 Figure 46: Silencing Hsp90... Phosphatidylinositol-3,4,5-trisphosphate PI3K PI3-Kinase PP2A Protein phosphatase 2A PTEN Phosphatase and Tensin Homolog Deleted on Chromosome 10 Ser Serine Cu/ZnSOD Cu/Zn Superoxide dismutase Thr Threonine xiii CHAPTER 1: INTRODUCTION 1. 1 Na+/ H+ exchanger- 1 (NHE1) 1. 1 .1 NHE1 isoforms and NHE1 structure The regulation of intracellular pH (pHi) is a tightly regulated parameter that has been shown to be instrumental for many cellular... family proteins Protein kinase B RhoA kinase Phosphatidylinositol 4,5bisphosphate Ribosomal S6 kinase Extracellular signalregulated kinase Nck-interacting kinase p38 mitogen-activated protein kinase Calmodulin Calmodulin Ca2+ calmodulindependent kinase II Calcineurin B homologous protein 1 Calcineurin B homologous protein 2 Calcineurin B homologous protein 3 Carbonic anhydrase II Heat shock protein 70 Death-domain-associated . LIST OF FIGURES ix LIST OF ABBREVIATIONS xii CHAPTER 1: INTRODUCTION 1 1. 1 Na + /H + exchanger- 1 (NHE1) 1 1. 1 .1 NHE1 isoforms and NHE1 structure 1 1. 1.2 Regulation of NHE1 3 1. 1.3. Physiological. protein of Hsp90 in MEF PTEN- /- cells 15 1 4 .1. 1 Increased stability of NHE1 protein in MEF PTEN- /- cells in comparison to MEF PTEN+ /+ cells 15 1 4 .1. 2 Hsp90 confers protection to NHE1 in MEF PTEN- /-. expression in MCF10CA1a 13 2 Figure 40: Akt 1 and 2 are detectable in MCF10CA1a 13 3 Figure 41: Silencing Akt induces downregulation of NHE1 protein expression in MCF10CA1a 13 3 Figure 42: NHE1-Akt interaction