1. p53-dependent Regulation of Mitochondrial Energy Production by the RelA NF-κB Subunit. Ini-Isabée Witzel 1,2, Renée Johnson 1,3 and Neil Perkins 1 1 Institute for Cellular &Molecular Biosciences, Newcastle University, UK. 2 New York University Abu Dhabi, Saadiyat Campus, Abu Dhabi, UAE. 3 Victor Chang Cardiac Research Institute, Sydney, Australia.

2. NF-κB Family Members Rel Homology Domain (RHD) (DNA binding and dimerisation) Transactivation N RelA (p65) c-Rel RelB C Proteolysis NF- κ B1 p105 p50 Ankyrin Repeats Proteolysis NF- κ B2 p100 p52

3. NF-κB and Disease NF-  B under control (mainly cytoplasmic) Normal cells Out of control NF-  B (aberrantly nuclear) Diseased cells Arthritis Asthma Atherosclerosis Inflammatory bowel diseases IIINFLAMMATORY DISEASES IIINFECTIONS AIDS Heliobacter pylori- associated gastritis Renal diseases Gut diseases Skin diseases Heart Failure Diabetes (type I & II) Ectodermal dysplasia Alzheimer’s diseases Incontinentia pigmenti CANCER All subunits have been implicated in cancer. NF-kB aberrantly active (nuclear): Leukemias Lymphomas Many solid tumours

4. Cancer Cells Self- sufficiency in growth signals Resistance to anti- growth signals Evasion of apoptosis Sustained angiogensis Limitless reproductive potential Tissue invasion and metatasis Abnormal metabolic pathways Immune system evasion Inflammation Unstable DNA and chromosome abnormality Cell. 2011 Mar 4;144(5):646-74. Hallmarks of Cancer

5. IKK NF-kB Cancer therapies (chemotherapeutic agents γ -irradiation) Stress ROI inducers (hypoxia) Carcinogens Tumour promoters (PMA, UV) Oncogene activation (Ras, Bcr-Abl) Genetic alteration (subunit expression, IkB deletion) Cancer-associated inducers of aberrant NF- κ B activity Cytokines Infection Inflammation (IL-1, TNF, chemokines) Angiogenesis (VEGF, TNF, IL-1, IL-8) Metastasis (ICAM-1, VCAM- 1, ELAM-1) Proliferation (Cyclin D1, c- Myc) Survival (Bcl-xL, XIAP, cIAP1 & 2) Immortality (telomerase) Tumour promotion (COX2, iNOS, MMP-9, uPA) Tumour promoting functions of NF-kB Is NF-κB also involved in alteration of cellular metabolism in cancer cells? NF- κ B and Cancer

6. RelA T505 Peptide (RelA): TTEPMLMEYPEAITRLVTGAQRPC Scramble Peptide (S): LATRQETGPIRMPEVYTAETMPLC 428521 Rel Homology Domain 1 19 551 301 RelA (p65) TA1 TA2 P T505 Fr 3-4 T505 Peptide Scramble WB: Mortalin kDa 191 97 64 51 39 28 19 14 Scramble Mortalin Fr 5-6 Fr 3-4 Fr 5-6 HeLa cells T505 Peptide Mortalin (mtHSP70) binds to the RelA T505 region Mutation of RelA at T505 impairs binding of RelA to Mortalin WB: RelA Mortalin mIgG IP: Null T505AT505D IP Inputs: Null RelA T505A T505D Mortalin mIgG Mortalin mIgG Mortalin mIgG Mortalin Mortalin binds to endogenous RelA in U-2 OS and 293 cells RelA reconstituted MEF cells RelA interacts with mitochondrial HSP70, Mortalin

7. Cytoplasm Mitochondria Null WB: RelA Mortalin VDAC  -tubulin BiP RelA T505A T505D Null RelA T505A T505D RelA reconstituted MEF cells MitochondriaCytoplasm U-2 OS WB: RelA Mortalin VDAC  -tubulin BiP Control Mortalin U-2 OS Control Mortalin siRNA U-2 OS cells RelA import into mitochondria is facilitated by an interaction with mortalin that is impaired by mutation of RelA at T505. RelA mitochondrial import is Mortalin dependent

8. O 2 consumption (average NRF) ** *** 0 0.5 1 1.5 siRNA: ControlRelAControlRelA Passage: Early Late U-2 OS cells 0 0.5 1 1.5 0 0.4 0.8 1.2 1.6 siRNA: Control RelA Control RelA O 2 consumption (average NRF) Treatment: Control Oligomycin ** *** Late Passage U-2 OS cells 0 0.6 1.2 1.8 ATP levels (average RLU / cell) siRNA: Control RelA Control RelA Treatment: Control Oligomycin ** Late Passage U-2 OS cells Late Passage U-2 OS cells 0 1 2 3 siRNA: Control RelA Glucose consumption (μg/min /50 000 cells) RelA-induced changes in ATP production have a physiological effect on the cells as indicated by phosphorylation of the energy sensing protein, AMPK. RelA regulates cellular energy production

9. p12 p20 p12 p20 Cytoplasm Mitochondria U-2 OS Cells RelA VDAC Mot α-tubulin RelA regulates mitochondrial gene expression Relative Enrichment (ChIP Assay) D-loop 0 1 2 3 Passage: Early Late Antibody: Control RelA Control RelA * 0 1 2 3 0 4 8 12 siRNA: Control RelA Control RelA Gene: D-loop Antibody: POLRMT * Relative Enrichment (ChIP Assay) Passage: Early Late Relative Cytochrome C Oxidase I mRNA Level Late Passage U-2 OS cells siRNA: Control RelA 0 0.04 0.08 0.12 * 0 0.02 0.04 0.06 0.08 siRNA: Control RelA Control RelA Average mRNA Abundance Gene: Cytochrome B Passage: Early Late ** *

10. Loss of p53: Results in reduced oxygen consumption and increased glycolysis 1 Reduced SCO2 and TIGAR and increased PGM and GLUT3 1,2 Enhances activation of NF-κB 2 Increased glycolysis in the absence of p53 via up- regulation of GLUT3 is RelA dependent 2 1. S. Matoba et al., Science 312, 1650 (Jun 16,2006). 2. K. Kawauchi, K. Araki, K. Tobiume, N. Tanaka, Nat Cell Biol 10, 611 (May, 2008) p53, RelA and Energy Production

11. Passage Number U-2 OS cells Phospho-RelA Thr505 Phospho-p53 Ser15 RelA p53 Actin 0 1 2 3 4 Relative enrichment (ChIP assay) Treatment: Control IPTG * Cytochrome B α-RelA H1299 wtp53 cells Induction of p53 reduces RelA binding to the mitochondrial genome 0 1 2 3 4 siRNA: Control RelA Control RelA O 2 consumption (average NRF) Treatment: Control IPTG * * * H1299 wtp53 cells MEF cells 0 0.4 0.8 1.2 Wild Type p53 -/- siRNA: Control RelA Control RelA ATP levels (average RLU /cell) ** * p53 mediates the switch in RelA regulation of cellular energy production RelA regulation of mitochondrial energy production is p53 dependent

12. p53 prevents RelA import into mitochondria by disrupting the interaction of RelA with mortalin IP : IPTG ControlIPTGInputs WB: RelA Mortalin ControlmIgGMortalinmIgGMortalin H1299 wtp53 cells WB: RelA VDAC Mortalin α-tubulin RSV P53-RSV RSV P53-RSV Cytoplasm Mitochondria H1299 cells RelA regulation of mitochondrial energy production is p53 dependent

13. Conclusions Early Passage (+p53) Nucleus Late Passage (-p53) p53 Glycolysis RelA

14. Acknowledgements Neil Perkins Renée Johnson Members of the Perkins Laboratory Members of CTP at NYUAD CRUK for funding our work!