Repression of the Protein Kinase PIM3 by an Mtorc1-Regulated Microrna
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Repression of the protein kinase PIM3 by an mTORC1-regulated microRNA The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:39987987 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Repression of the protein kinase PIM3 by an mTORC1-regulated microRNA A dissertation presented by Ilana Ashley Kelsey to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts August 2017 © 2017 Ilana Ashley Kelsey All rights reserved. Dissertation Advisor: Brendan Manning Ilana Ashley Kelsey Repression of the protein kinase PIM3 by an mTORC1-regulated microRNA Abstract The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that is often aberrantly activated in cancer. However, mTORC1 inhibitors, such as rapamycin, have limited effectiveness as single agent cancer therapies, with feedback mechanisms inherent to the signaling network thought to diminish the anti-tumor effects of mTORC1 inhibition. The goals of this dissertation were to characterize pro-survival effectors activated upon mTORC1 inhibition, and to determine the functional significance of these downstream targets, including relevance to the development of targeted therapies in combination with mTORC1 inhibitors. I identify the repression of protein kinase and proto-oncogene PIM3 downstream of mTORC1 signaling. PIM3 expression is suppressed in cells with loss of the tuberous sclerosis complex (TSC) tumor suppressors, which exhibit growth factor-independent activation of mTORC1, and in the mouse liver upon feeding-induced activation of mTORC1. Inhibition of mTORC1 with rapamycin induces PIM3 transcript and protein levels in a variety of settings. Suppression of PIM3 involves the sterol regulatory element-binding (SREBP) transcription factors SREBP1 and 2, whose processing and mRNA expression are stimulated by mTORC1 signaling. I found that PIM3 repression is mediated by miR-33, an intronic microRNA encoded within the SREBP loci, the expression of which is decreased with rapamycin. I sought to better understand the functional implications of miR-33 induction by mTORC1, and the subsequent induction of PIM3 upon mTORC1 inhibition. Specifically, I show that PIM inhibition in combination with mTOR inhibitors may be a promising therapy in some cancer settings. I also identify several additional mTORC1-regulated miR-33 targets that contribute to cell survival and metabolism, iii including PIM1, which is closely related to PIM3. Finally, I explore the metabolic changes affected by PIM inhibition, providing an additional rationale for the regulation of PIM3 by mTORC1. Collectively, these studies identify a pro-survival kinase that is activated upon mTORC1 inhibition while highlighting the importance of further characterization of miR-33 targets altered downstream of mTORC1. Our results will guide future studies of mTORC1-regulated microRNAs and pro-survival pathways, with potential implications for the effects of mTORC1 inhibitors in TSC, cancer, and the many other disease settings influenced by aberrant mTORC1 signaling. iv TABLE OF CONTENTS ABSTRACT iii LIST OF FIGURES viii GLOSSARY OF TERMS x DEDICATION xv ACKNOWLEDGEMENTS xvi CHAPTER 1: INTRODUCTION 1 1.1 mTORC1 is a major regulator of cell growth and metabolism 1.1.1 Overview 1.1.2 Upstream regulation of mTORC1 1.1.3 Downstream processes regulated by mTORC1 1.1.4 mTORC1 control of coding and non-coding RNA expression 1.1.5 mTORC1 signaling in disease 1.2 The PIM kinase family in signaling and disease 1.2.1 Overview 1.2.2 Upstream regulation of PIM kinases 1.2.3 Downstream targets of PIM kinases 1.2.4 PIM kinases in cancer 1.3 Specific Aims and Overview of the Dissertation 1.4 References v CHAPTER 2: mTORC1 SUPPRESSES PIM3 EXPRESSION VIA miR-33 ENCODED BY THE SREBP LOCI 50 2.1 Abstract 2.2 Introduction 2.3 Materials and Methods 2.4 Results 2.4.1 PIM3 expression is repressed downstream of mTORC1 and induced by mTOR inhibitors 2.4.2 A survey of mTORC1-regulated transcription factors identifies SREBP1 and 2 as upstream of PIM3 2.4.3 miR-33, an intronic microRNA within the SREBP loci, targets PIM3 downstream of mTORC1 2.5 Discussion 2.6 Acknowledgements 2.7 Author Contributions 2.8 References CHAPTER 3: BIOLOGICAL SIGNIFICANCE OF miR-33 INDUCTION AND PIM REPRESSION BY mTORC1 75 3.1 Abstract 3.2 Introduction vi 3.3 Materials and Methods 3.4 Results 3.4.1 miR-33-targeted transcripts are changing downstream of mTORC1 3.4.2 Additive effects of dual inhibition of mTORC1 and PIM kinases in Tsc2-/- MEFs 3.4.3 Implications for PIM effects on metabolism, particularly cellular NAD+ levels 3.5 Discussion 3.6 Acknowledgements 3.7 References CHAPTER 4: CONCLUSIONS 102 4.1 Overview 4.2 Challenges of targeting the mTORC1 signaling axis in disease 4.2.1 Overview 4.2.2 Crosstalk and feedback pathways contribute to rewiring of the signaling network in response to specific inhibitors 4.2.3 Redundancies in the greater mTORC1 network enable sustained signaling 4.2.4 Resistance to targeted therapies is intrinsic to the wiring of the network 4.3 Future directions 4.4 References vii LIST OF FIGURES FIGURE 1.1 – Regulation of mTORC1 by a variety of upstream inputs 4 FIGURE 1.2 – Amino acid sensing and mTORC1 activation at the lysosome 7 FIGURE 1.3 – mTORC1 stimulates anabolic pathways to support cell growth and proliferation 9 FIGURE 1.4 – mTORC1 controls a vast transcriptional program in order to regulate anabolic 13 processes and stimulate cell growth FIGURE 1.5 – microRNA regulation downstream of mTORC1 16 FIGURE 1.6 – A wide network of transcription factors regulates the expression of the PIM kinases 20 FIGURE 1.7 – The PIM kinases promote cell survival through a variety of pathways 24 FIGURE 1.8 – The PIM kinases stimulate Myc expression and transcription at multiple steps 27 FIGURE 2.1 – PIM3 is repressed downstream of mTORC1 57 FIGURE 2.2 – PIM3 repression by mTORC1 is observed in a variety of human cancer settings 59 FIGURE 2.3 – Identification of the mTORC1 effectors SREBP1 and 2 as being upstream of 61 PIM3 regulation FIGURE 2.4 – PIM3 is induced upon inhibition of SREBP1 and 2 63 FIGURE 2.5 – An SREBP-intronic microRNA, miR-33, targets PIM3 expression downstream 64 of mTORC1 FIGURE 3.1 – miR-33 targets are induced by rapamycin 81 FIGURE 3.2 – Combined inhibition of PIM3 and mTORC1 has an additive effect in MEFs 83 FIGURE 3.3 – Metabolomic profiling in high/low PIM3 settings reveals changes in NAD+ levels 86 and synthesis upon PIM inhibition FIGURE 3.4 – Supporting metabolomics data for Figure 3.3 88 viii FIGURE 4.1 – Graphical summary of the dissertation 104 FIGURE 4.2 – Feedback pathways in the mTORC1 signaling network 106 FIGURE 4.3 – Pathway convergence on shared effectors 110 ix GLOSSARY OF TERMS 25-HC 25-hydroxycholesterol 4E-BP eIF4E binding proteins ABCA1 ATP-binding cassette transporter A1 AMP adenosine monophosphate AMPK AMP kinase ATF4 activating transcription factor 4 ASK1 apoptosis signaling kinase 1 ATG7 autophagy-related 7 C/EBP-α CCAAT/enhancer-binding protein-α CAD carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase CDC25A/C cell division cycle 25 homolog A and C C-TAK1 Cdc25 C-associated kinase 1 DEPTOR DEP domain-containing mTOR-interacting protein eEF2K eukaryotic elongation factor 2 kinase EGF epidermal growth factor EGFR EGF receptor eIF4B eukaryotic translation initiation factor 4B eIF4E eukaryotic translation initiation factor 4E ER endoplasmic reticulum ERK/MAPK mitogen-activated protein kinase FAS fatty acid synthase FBS fetal bovine serum FKBP12 FK506 binding protein of 12 kDa FOXO forkhead box O x GAB1/2 GRB2-associated binder 1 and 2 GAP GTPase-activating protein GEF guanine nucleotide exchange GRB10 growth factor receptor bound protein 10 GSK3 glycogen synthase kinase 3 HCC hepatocellular carcinoma HIF-1α hypoxia-inducible factor alpha HK2 hexokinase 2 HOXA9 homeobox protein A9 HSP90 heat shock protein 90β IGF1 insulin-like growth factor 1 IR insulin receptor IRS-1/2 insulin receptor substrates 1 and 2 INSIG insulin-induced gene JNK c-Jun N-terminal kinase KLF5 Kruppel-like factor 5 LAM lymphangioleiomyomatosis LC-MS/MS liquid chromatography-mass spectrometry/mass spectrometry Lipin1 phosphatidic acid phosphatase LPIN1 MAX Myc-associated factor X MEF mouse embryonic fibroblast MEK MAPK kinase mLST8 mTOR-associated protein, LST8 homologue mRNA messenger RNA MTHFD2 methylene tetrahydrofolate dehydrogenase 2 mTOR mechanistic target of rapamycin xi mTORC1 mTOR complex 1 mTORC2 mTOR complex 2 MuLV Moloney murine leukemia virus NADH nicotinamide adenine dinucleotide nCoR1 nuclear receptor co-repressor 1 NF1 neurofibromin 1 NF-Κb nuclear factor- Κb Nrf1 nuclear factor, erythroid 2-like 1 p21Cip1/WAF1 cyclin dependent kinase inhibitor 1A p27KIP1 cyclin dependent kinase inhibitor 1B PA phosphatidic acid Pax-5 paired box gene 5 PDCD4 programmed cell death 4 PDGF platelet-derived growth factor PDGFR PDGF receptor PDK1 phosphinositide-dependent kinase-1 PGC1α peroxisome proliferator-activated receptor gamma coactivator-1 PI propidium iodide PI3K phosphoinositide 3-kinase PIM proviral