p53 and Apoptosis: Master Guardian and Executioner The Biology of Cancer Chapter 9: p53 and Apoptosis: Master Guardian and Executioner

Why p53? Need to eliminate malfunctioning cells Must have a decision maker (p53) If too much damage, initiates programmed cell suicide (Apoptosis) P53 is dangerous for the cancer cell because its job is to kill cancer cell

Discovery of p53 Originally identified as an oncogene in association with Large T antigen in SV40 Over-expressed in murine tumors Forms found in tumors are mutant, have to amino acid changes in functional domains Does not conform to Knudson’s “two hit hypothesis” model

How can a mutant form foster tumor growth? Yeast: Mutant alleles of gene can interfere with wild-type gene function “Dominant-interfering” or “Dominant-Negative” Wild-type P53 found to be a homo-tetramer (made from 4 identical units) Single mutation produce normal and error copies of each unit in equal proportion

Figure 9.7a The Biology of Cancer (© Garland Science 2007)

Out of 16 tetramers, only one will function normally Figure 9.7b The Biology of Cancer (© Garland Science 2007)

What happens when there is a mutation in p53? Mutant form makes tetramer but it cannot function normally P53 efficiency reduced to 1/16 th of normal Advantage for tumors Many tumors also have LOH at mutation locus for p53 They cannot tolerate even 1/16 efficiency of p53

Figure 9.4 The Biology of Cancer (© Garland Science 2007)

Variation in type of mutation seen in different genes in cancer Figure 9.6a The Biology of Cancer (© Garland Science 2007)

Most p53 mutations are in the DNA binding domain Figure 9.6b The Biology of Cancer (© Garland Science 2007)

P53 has a short half-life Cycloheximide blocks protein synthesis When added to cells with wild-type p53 in-vitro, amount of p53 protein decayed with ½ life of 20 minutes p53 has a “futile” cycle Useful way to create a “fast switch” for a critical protein. Turns on immediately when its degradation is inhibited.

What activates p53? X-rays, UV radiation damage DNA damaging chemo agents Inhibitors of DNA synthesis Microtubule disruption Many carcinogens, drugs Hypoxia High Nitrous Oxide concentrations

More activation signals Oncogene upregulation Changes in methylation of chromosomes Acidified growth medium Reduction in ribonucleotides Blocks in RNA/DNA synthesis

P53 causes “growth arrest” if it senses damage Initiates a “Repair” program If damage persists or repair fails, initiates cell suicide Figure 9.8 The Biology of Cancer (© Garland Science 2007)

How p53 stops cell cycle X rays upregulate p53 which induces p21Cip1 p21 binds to Cyclin D, CDK4/6 X-rays do not induce p21 when p53 mutated Direct correlation seen in p53 levels and apoptotic rate for different amounts of X-ray Figure 9.9 The Biology of Cancer (© Garland Science 2007)

P53 protein is transcription factor for MDM2 p53/MDM2 “futile” cycle P53 protein is transcription factor for MDM2 MDM2 ubiquitylates p53 for degradation in proteasome Figure 9.11 The Biology of Cancer (© Garland Science 2007)

P53 protein recognizes a specific DNA sequence: Pu-Pu-Pu-C-A/t-T/a-G-Py-Py-Py repeated twice with a gap of 0-13 MDM2 binds p53 protein in a small N-terminus region Grey area allows it to form a tetramer Red: NLS or Nuclear Localization Signals + amino acid for DNA binding “Proline rich” region contributes its apoptotic activity Figure 9.12 The Biology of Cancer (© Garland Science 2007)

p53 activation mechanism Double stranded DNA breaks (X-rays) ATM ATR Kinase  phosphorylation of p53 to stabilize it DNA damaging agents ATM CKII phosphorylate p53 Aberrent Growth signals e.g. (pRb-E2F) disregulation: Mechanism ? Hypoxia: Mechanisms?

P53 stabilized by phosphorylation Phosphorylation done by kinases like ATM, Chk1, Chk2 which are activated by DNA damage They alter the p53 domain recognized by MDM2 ATM can also phosphorylate MDM2 to deactivate it ATM  p53 ATM --| MDM2 MDM2 --| p53 Figure 9.13 The Biology of Cancer (© Garland Science 2007)

MDM2 is a “funny” oncogene Acts by “antagonizing” a tumor suppressor Prevents entry into cell cycle arrest, senescence, cell suicide MDM2 is under control of other proteins: E.g. Ras Raf  MapK  ETS/AP-1 which through FOS + Jun transcription factors can increase MDM2 levels SNP 309 in MDM2 intron is cancer promoting

P14ARF ARF = Alternate Reading Frame Transcriptional promotor 13 kb upstream of gene for p16INK4A deletes exon 1 and an alternate reading frame in Exon 2 Result = ARF mRNA Figure 9.14 The Biology of Cancer (© Garland Science 2007)

ARF is a tumor suppressor Shuts down MDM2 by sequestering it in nucleolus ARF + p53 monitor intracellular signalling and cell cycle pRb --| E2F  ARF --| MDM2 --| p53  death p14ARF has an E2F recognition sequence Elimination of ARF removes check on E2F levels

Figure 9.15b The Biology of Cancer (© Garland Science 2007)

Table 9.2 The Biology of Cancer (© Garland Science 2007)

P53 is not involved in “routine” cell death e.g. in morphogenesis Mouse hand morphogenesis. Black dots are cells undergoing apoptosis Figure 9.19 The Biology of Cancer (© Garland Science 2007)

Li-Fraumini Syndrome Predisposition to Glioblastomas, Leukemias, Breast, Lung, Pancreatic tumors Mandelian inheritance of trait Traced to scattered mutations in p53 gene on Chr17-p13 Figure 9.20 The Biology of Cancer (© Garland Science 2007)

The bcl-2 Oncogene B-Cell Lymphoma Gene seen to be upregulated in tumors Mutation is reciprocal translocaton in Chr14/Chr18 Mutant bcl-2 inserted in mouse germline and expressed in lymphocyte precursor has no effect on survival Myc upregulation + bcl-2 mutation killed mice in < 2 months Myc upregulation is also potent killer but not as potent as Myc + bcl-2 Figure 9.22b The Biology of Cancer (© Garland Science 2007)

Mechanism of action of bcl-2 Mutant bcl-2 prolongs survival of lymphocytes in-vitro In vivo, prolongs lymphocyte life but cells are not proliferating  no tumors induced Bcl-2 seems to act in the “opposite way” from p53, causing a halt in “death” program

Figure 9.27c The Biology of Cancer (© Garland Science 2007)

Apoptosis and bcl-2 bcl-2 is anti-apoptotic Found localized in outer membrane of mtDNA ! Apoptosis is triggered by depolarization of outer membrane of mtDNA and release of Cytochrome C Cytochrome C associates with other proteins to initiate cell death

Apoptosis = “Programmed Cell Death” Figure 9.29 The Biology of Cancer (© Garland Science 2007)

Two apoptotic pathways P53 induced – driving genes like Bax which open mtDNA channels Extracellular, initiated by signals from transmembrane “death receptors”

There is a third pathway T-cell and NK cell mediated Immune system Killer cells attach a protease (like a bomb) to cell outer membrane. When internalized it cleaves and activates procaspase 3,8,9  Cell death

Convergence of Intrinsic and Extrinsic Apoptotic pathways Figure 9.32 The Biology of Cancer (© Garland Science 2007)

P53 can also use the extrinsic pathway by inducing a gene for the Fas “death” receptor inducing IGFBP-3 which binds to extracellular IGF-1,2 ligands to reduce effect of external anti-apoptotic signals Figure 9.33 The Biology of Cancer (© Garland Science 2007)

Anti-Apoptotic Strategies of Cancer Cells Point mutations in p53 to disable pathways Deletion or promotor methylation of ARF Overexpression of MDM2 Mis-localization of p53 (sequestering in cytoplasm) Hijacking of apoptotic pathway (Melanoma) Inactivating Bax (Colon Cancer) Disregulating bcl-2 (Follicular Lymphoma) Hyperactivation of PI3K  Aft  PkB pathway Activation of NF-kB pathway

Anti-apoptotic Pathway: Figure 9.34 The Biology of Cancer (© Garland Science 2007)

Table 9.5 The Biology of Cancer (© Garland Science 2007)

Different post transcriptional modifications of p53 protein Figure 9.35 The Biology of Cancer (© Garland Science 2007)

Autophagy or Cell Self-Cannibalism A survival mechanism. Is it a mechanism for therapy? Beclin-1 induces autophagy and is down-regulated by tumors Figure 9.36 The Biology of Cancer (© Garland Science 2007)

The p53 circuit board Figure 9.37 The Biology of Cancer (© Garland Science 2007)