Apoptosis Tumorsuppressor genes function like brakes keep cell

Apoptosis

Tumor-suppressor genes, function like brakes, keep cell numbers down, either by inhibiting progress through the cell cycle and thereby preventing cell birth, or by promoting programmed cell death (also called apoptosis). When cellular tumor suppressor genes are rendered non-functional through mutation, the cell becomes malignant. Examples are the gene encoding the retinoblastoma protein (Rb), inactivated in retinoblastomas, p 53, and p 16 INK 4 a, which inhibits cyclin-dependent kinases and is inactivated in many different tumors.

Oncogenes stimulate appropriate cell growth under normal conditions, as required for the continued turnover and replenishment of the skin, gastrointestinal tract and blood, for example. Cells with mutant oncogenes continue to grow (or refuse to die) even when they are receiving no growth signals. Examples are Ras, activated in pancreatic and colon cancers, and Bcl-2, activated in lymphoid tumours. Amplification of oncogenes (more than their normal gene copy number) is also found in cancer: MDM 2 is amplified in liposarcomas.

Roles of p 53 in apoptosis 1. p 53 induces apoptosis through transcriptional activation of proapoptotic genes, such as Puma, Noxa, p 53 AIP 1, Bax, Apaf-1 etc. 2. It can also directly induce apoptosis by localizing to mitochondria via interaction with Bcl-2 family protein Bcl -x. L and facilitating Bax oligomerization Reading: Vousden and Lu: Nature Reviews Cancer, 2002, 2: 594 -604.

p 53 and of apoptosis Ref: Mol. Cell, 2003, 11(3): 552 -4

p 53 and apoptosis Ref: Cell, 2002, 108: 153 -164

Tumor Suppressor p 53 • First identified as a protein associated with viral oncogenes • Mutated/inactivated in a majority of human cancers • Integrates numerous signals that control cell life and death • A common denominator in human cancer • Understanding functions and regulation of p 53 is of great importance in cancer biology and cancer therapy

The p 53 pathways

The p 53 network

p 53 is a sequence-specific DNA binding protein 1. p 53 central core-domain interacts directly with DNA 2. p 53 binding sites consist of four copies of the pentamer consensus sequence Pu. Pu. C(A/T). The pentamers are oriented in alternating directions. A short stretch of sequence up to 13 bp may be inserted between the pairs of pentamers. The p 53 target genes in the human genome usually carry the consensus sequence. 3. Amino acid residues in the core-domain that are critical for DNA-binding are among the “hot-spots” of tumor-derived p 53 mutations, attesting to the importance of DNA-binding for p 53’s tumor suppression function.

Structure of p 53 core-domain Ref: Science, 265: 346 -355, 1994

p 53 and apoptosis Apaf-1 p 53 AIP 1

p 53 in apoptosis 1. p 53 mediates apoptosis in response to DNA damage, oncogene expression (adenovirus E 1 A, myc etc. ), or withdrawal of growth factors 2. Overexpression wild-type of p 53 leads to apoptosis 3. p 53 can induce the expression of proapoptotic genes, such as Bax (ref Cell, 80: 293) and p 53 AIP 1 (ref: Cell, 102: 849)

p 53 in apoptosis 4. p 53 can also repress transcription of certain genes, and it has been proposed that the repression function may also be required for apoptosis (ref: Genes Dev 13: 2490 -501) 5. In vivo, p 53 transactivation mutant is defective in inducing apoptosis, at least for some cell types (ref: EMBO J. 19: 4967 -4975)

Physiological relevance of p 53 -induced apoptosis • Suppress oncogene-induced transformation • Inhibit tumor growth and progression • Remove cells with severe DNA damage • Effectiveness of cancer chemotheraphy correlates with the ability to induce p 53 -dependent apoptotic response

Tumor-derived mutations affecting apoptosis Protein Role in apoptosis Ref p 53 Mutated or altered expression in many cancers. Vogelstein et al. , Initiates the intrinsic apoptotic pathway. p 53 -/ 2000 - cells are resistant to drug induced apoptosis. p 19 ARF Mutated or altered expression in many cancers. Sherr and Weber, Blocks MDM 2 inhibition of p 53. Enhances 2000 drug-induced apoptosis by p 53. Rb Mutated in some cancers, and functionally disrupted in many cancers. Inhibits E 2 Fmedidated transcription. Loss of Rb function induces p 53 -dependent and independent apoptosis. Harbour and Dean, 2000 Chk 2 Mutated in Li-Fraumeni syndrome. Senses DNA double strand breaks and phosphorylates and stabilizes p 53. Khanna and Jackson, 2001 Ref: Cell, 2002, 108: 153 -164

Tumor-derived mutations affecting apoptosis Protein Role in apoptosis Ref ATM Mutated in ataxia-talangiectasia syndrome. Senses DNA double strand breaks and stabilizes p 53. Deficiencies increase risk of developing haematological malignancies and breast cancer Khanna and Jackson, 2001 Bax (p 53 Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage. Sufficient but not necessary for drug-induced apoptosis. Rampino et al. , 1997 Bak Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage. Sufficient but not necessary for drug-induced apoptosis. Kondo et al. , 2000 PTEN (p 53 Mutated or altered expression in cancers. Regulates Di Cristofano and Akt activation and subsequent phosphorylation of Pandolfi, 2000 Bad. Loss of PTEN results in resistance to many apoptotic stimuli. target gene) Ref: Cell, 2002, 108: 153 -164

Tumor-derived mutations affecting apoptosis Protein Role in apoptosis Ref Apaf-1 (p 53 target gene) Mutated and transcriptionally silenced in Soengas et al. , melanoma and leukemia cell lines. Necessary for 2001 activation of caspase-9 following cytochrome c release. Apaf-1 -/- cells are chemoresistant. CD 95/Fas Mutated and down-regulated in lymphoid and solid tumors. Initiates the extrinsic apoptotic pathway. Loss of function is associated with resistance to drug-induced cell death. TRAILR 1/R 2 Caspase-8 Mutated in metastatic breast cancers. Initiate the Shin et al. , 2001 extrinsic apoptotic pathway. Mutations lead to suppression of death receptor-mediated apoptosis. Muschen et al. , 2000 Gene silenced in neuroblastomas. Activates both Teitz et al. , 2000 extrinsic and intrinsic apoptotic pathways. Silencing results in resistance to drug-induced apoptosis. Ref: Cell, 2002, 108: 153 -164

Tumor-derived mutations affecting apoptosis Protein Role in apoptosis Ref Bcl 2 Frequently overexpressed in many tumors. Antagonises Bax and/or Bak and inhibits mitochondrial membrane disruption. Inhibits drug-induced apoptosis. Reed, 1999 MDM 2 Overexpressed in some tumors. Negative regulator of p 53. Inhibits drug-induced p 53 activation. Sherr and Weber, 2000 IAPs Frequently overexpressed in cancer. Down regulation of XIAP induces apoptosis in chemoresistant tumors. Deveraux and Reed, 1999 NFk. B Deregulated activity in many cancers. Baldwin, 2001 Transcriptionally activates expression of antiapoptotic members of the Bcl-2 and IAP families. Can inhibit both the extrinsic and intrinsic death pathways and induce drug-resistance. Ref: Cell, 2002, 108: 153 -164

Tumor-derived mutations affecting apoptosis Protein Role in apoptosis Ref Myc Deregulated expression in many cancers. Induces proliferation in the presence of survival factors, such as Bcl-2, and apoptosis in the absence of survival factors. Can sensitise cells to drug-induced apoptosis. Evan and Vousden, 2001 Akt Frequently amplified in solid tumors. Phosphorylates Datta et al. , 1999 Bad. Hyperactivation induces resistance to a range of apoptotic stimuli including drugs. PI 3 K Overexpressed or deregulated in some cancers. Responsible for activation of Akt and downstream phosphorylation of Bad. Inhibition of PI 3 K enhances chemotherapeutic drug-induced apoptosis. Roymans and Slegers, 2001 Ras Mutated or deregulated in many cancers. Activates PI 3 K and downstream pathways. Induces proliferation and inhibits c-myc and drug-induced apoptosis. el-Deiry, 1997 Ref: Cell, 2002, 108: 153 -164

Tumor-derived mutations affecting apoptosis Protein Role in apoptosis Ref FLIP Overexpressed in some cancers. Prevents Tepper and Seldin, activation of caspase-8 and apoptosis induced by 1999 some chemotherapeutic drugs. Ref: Cell, 2002, 108: 153 -164

p 53 directly mediates mitochondrial mechanism of apoptosis 1. Early studies indicate that transactivation-defective mutants of p 53 are capable of inducing apoptosis, implying a transcription-independent role for p 53 in apoptosis. 2. DNA-damage leads to mitochondrial translocation of p 53. 3. p 53 binds to Bcl-2 family protein Bcl-x. L to influence cytochrome c release. 4. p 53 directly activates the proapoptotic Bcl-2 protein Bax in the absence of other proteins to permeabilize mitochondria and engage the apoptotic program. 5. p 53 can release both proapoptotic multidomain proteins and BH 3 -only proteins [Proapoptotic Bcl-2 family proteins that share only the third Bcl-2 homology domain (BH 3)] that are sequestered by Bcl-x. L. Ref: Mol. Cell, 2003, 11: 577 -90; Science, 2004, 303: 1010 -4.

How p 53 functions in mitochondria to induce cell death 1. p 53 or Bax alone does not permeabilize membrane, but they together can do so. 2. p 53 facilitates Bax oligomerization. 3. p 53 binds to Bcl-x. L, but not to Bax. 4. p 53 -Bcl-x. L interaction releases Bax. 5. Released Bax forms oligomers in mitochondrial membrane, leading cytochrome c release and apoptosis. 6. The proline-rich domain (aa 62 -91 in mouse) of p 53 is required for this effect. Ref: Science, 2004, 303: 1010 -4.

Viral oncogenes inhibit p 53 functions 1. p 53 was first identified as binding protein of SV 40 large T antigen in 1979, which interacts with p 53 core domain 2. Adenovirus oncoprotein E 1 B 55 -k. Da binds to p 53 and inhibits p 53 transactivation activity 3. HPV E 6 binds to p 53 and targets it for ubiquitin-mediated degradation.

Using p 53 to kill cancer cells The p 53 protein is a tumor suppressor — it keeps cell numbers down by stopping cells from multiplying or by promoting cell death. Loss of p 53 occurs in most human cancers, so it would be useful to be able to restore its function. Several innovative strategies have been suggested: • Introduce normal p 53 genes into a cancer cell with mutant p 53. • Introduce a small compound that converts mutant p 53 proteins from an abnormal to a normal shape. • Add a protein that attaches itself to mutant p 53 and kills cells. • Stimulate the host's immune response to mutant p 53 peptides.

Using p 53 to kill cancer cells • Introduce drugs that disrupt the interaction between the MDM 2 or E 6 proteins and p 53. (MDM 2 and E 6 negatively regulate p 53; they are present at abnormally high levels in some cancer cells, so 'quench' any normal p 53. ) • Introduce the adeno-associated virus, which mimics damaged DNA. Cells with mutant p 53 cannot activate the usual p 53 -dependent 'checkpoint' that is induced by DNA damage, and eventually die. • Infect cells with viruses that can replicate only in cells without normal p 53; the viruses kill these

p 53 in DNA repair and apoptosis ROS: reactive oxygen species Ref: Bensaad & Vousden, Nature Med,

Summary 1. p 53 is a tumor-suppressor protein that induces apoptotic cell death in response to oncogenic stress. Malignant progression is dependent on loss of p 53 function, either through mutation in the TP 53 gene (which encodes p 53) itself or by defects in the signaling pathways that are upstream or downstream of p 53. 2. Mutations in TP 53 occur in about half of all human cancers, almost always resulting in the expression of a mutant p 53 protein that has acquired transforming activity. 3. p 53 -induced apoptosis depends on the ability of p 53 to activate gene expression. 4. p 53 can also directly trigger the apoptotic response, by interacting with Bcl-2 family protein. 5. The apoptotic and cell-cycle arrest activities of p 53 can be separated, and apoptotic cofactors that play a specific role in allowing p 53 -induced death are being identified. Ref: Nature Reviews Cancer, 2, 594 - 604 (2002)

Summary 6. Phosphorylation of p 53 regulates its ability to activate the expression of apoptotic target genes, and other posttranslational modifications such as acetylation might also have a role. 7. In tumors that retain wild-type p 53, the apoptotic response might be hindered by defects in the apoptotic cofactors. These, therefore, represent additional targets for the design of therapeutics that are aimed at reactivating p 53 -mediated apoptosis in cancer cells. Ref: Nature Reviews Cancer, 2, 594 - 604 (2002)