2. Prosurvival mechanisms I. Endogenous prosurvival mechanisms II. Autophagy III. Induced prosurivival mechanisms MECHANISMS OF CARCINOGENESIS SPRING SEMESTER (Dean Tang, April 27, 2011)

3. Two Apoptotic Pathways

4. Prosurvival mechanisms: BH3-only proteins are tightly regulated 1.Like Egl-1, some BH3-only proteins (e.g., Puma, Noxa, Hrk, and Bim) are normally not expressed in some cell types and need transcriptional activation. 2.Some BH3-only proteins are expressed but are not lethal. For example, Bim in many cells is expressed but remains bound to tubulin or tubulin-based dynein motor complexes and therefore is not proapoptotic. Bid is expressed in many cells but generally needs a post-translational cleavage to generate proapoptotic tBid. Bad is abundant in many cells but inactive due to PHOSPHORYLATION and sequestration by 14-3-3 proteins. BH3-only BCL-2 BH1-3 pro-apoptotic proteins adaptorcaspaseanti-apoptotic Bcl-2 proteins Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa

5. BAD: Phosphorylation sequesters it in the cytosol

6. *Phosphotidylinositol 3-kinase, or PI3K, is activated by multiple mitogens and survival factors. Prototypical examples include: NGF in neuronal cells, IL-3 and other cytokines in lymphocytes, PDGF and EGF in mesenchymal cells, and IGF-1 in epithelial cells. *These tyrosine kinase receptors, as well as seven transmembrane G protein-coupled receptors and activated Ras, recruit PI3K to the membrane, which then catalyzes the transfer of phosphate from ATP to the D3 position of the inositol ring of membrane-localized phosphoinositides. *The main products, PI3,4P and PI3,4,5P, function as signaling molecules that can activate TK, small G proteins, and ser/thr kinases such as atypical PKCs, pp70 S6K, and c-Akt. Activation of c-Akt is the major survival function. A Major Prosurvival Pathway: PI3K-Akt

7. *Akt (PKB) has a central kinase domain with ser/thr substrate specificities, an amino terminal PH (which mediates protein-lipid and/or protein-protein interactions), and a C-terminal hydrophobic and proline-rich domain. *Direct binding of PI3,4,5P or related lipids to the PH domain in Akt leads to its translocation to plasma membrane. At this location and with PIP bound, Akt is phosphorylated (at Thr308 and Ser473) and activated by PKDs (3-phosphoinositide-dependent protein kinases). Integrin-linked kinase (ILK) is also a PIP-dependent Akt kinase (Ser473). *Activated Akt has three main apoptosis-related targets: Bad, caspase-9, and forkhead (FKH) transcription factors. Phosphorylation of Bad (at Ser136) and of caspase-9 inactivate these pro- apoptotic proteins. Phosphorylation of FKH and Bad sequesters them in the cytosol by binding to 14-3-3. *Transfection with constitutively active Akt blocks apoptosis induced by deprivation, UV irradiation, matrix detachment, cell-cycle disruption, DNA damage, TGF , anti-Fas, etc. *Conversely, trasnfection with dominant-negative alleles of Akt (catalytically inactive Akt, a mutant Akt consisting only of the PH domain) blocks the pro-survival functions of survival factors. *PTEN is a lipid phosphatase that dephosphorylates PI3K-generated 3’-phosphorylated phosphotidylinositides. *Mitogens and survival factors activate a parallel survival pathway: Raf-MEK-MAPK pathway. Ras-dependent survival is mediated by both PI3K and Raf-MEK-MAPK pathway.

8. Prosurvival mechanisms: BH3-only proteins are tightly regulated 1.Like Egl-1, some BH3-only proteins (e.g., Puma, Noxa, Hrk, and Bim) are normally not expressed in some cell types and need transcriptional activation. 2.Some BH3-only proteins are expressed but are not lethal. For example, Bim in many cells is expressed but remains bound to tubulin or tubulin-based dynein motor complexes and therefore is not proapoptotic. Bid is expressed in many cells but generally needs a post-translational cleavage to generate proapoptotic tBid. Bad is abundant in many cells but inactive due to PHOSPHORYLATION and sequestration by 14-3-3 proteins. 3.Bim undergoes biallelic deletion in mantle cell lymphoma and promoter methylation in other B-cell lymphomas. A few human lymphomas also have Noxa gene mutations. Bik/Nbk is mutated in renal cell carcinomas. BH3-only BCL-2 BH1-3 pro-apoptotic proteins adaptorcaspaseanti-apoptotic Bcl-2 proteins Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa

9. BH3-only proteins may have physiological/prosurvival functions *BID is required for myeloid homeostasis: Bid-deficient mice, as they age, spontaneously develop a myeloproliferative disorder resembling CMML (chronic myelomonocytic leukemia). *Bid plays a role in DNA-damage response and in promoting cell survival (Zinkel SS, et al., Cell 122, 579-591, 2005; Kamer I et al., Cell 122, 593-603, 2005). *Bad is involved in normal glycolysis and in promoting cell survival (Danial NN et al., Nature 424, 952-956, 2003; Seo et al., JBC 279, 42240-42249, 2004). *Most bim-deficient mice die embryonically, suggesting Bim may also have a normal physiological function. Bim may have a role in regulating cytoskeletal integrity. *bid -/- mice (Yin et al., Nature 400:886-891, 1999.) --- Animals develop normally --- Resistant to anti-Fas induced liver failure *bim -/- mice (Bouillet et al., Science 286, 1735-1738, 1999.) --- A significant number of bim-null, and even some of the bim +/- mice die in utero prior to E9.5 for unknown reasons. --- Bim deficiency causes an SLE (systematic lupus erythematosus)- like autoimmune disease.

10. Anti-apoptotic Bcl-2 proteins function as endogenous prosurvival molecules 1.Bcl-2 and Bcl-xL are indispensable for stem cell development and are critical for the survival of stem cells and many mature cell types. Bcl-2 and/or Bcl-xL are overexpressed perhaps in all tumor cells. 2.Mcl-1 is critical for the survival of HSCs, neutrophils (not macrophages), fibroblasts, keratinocytes, and many other cell types. Mcl-1 is also overexpressed in several cancers. 3.Bcl-w is expressed in a majority of invasive gastric adenocarcinomas. BH3-only BCL-2 BH1-3 pro-apoptotic proteins adaptorcaspaseanti-apoptotic Bcl-2 proteins Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa

11. *bcl-2 -/- mice (Nakayama et al., Science 261: 1584, 1993; Veis et al., Cell 75:229-240, 1993) --- Defects in renal development (essential for kid. stem cells) --- Premature loss of B/T lymphoid cells: Immunodeficiency --- Defects in melanocyte development (essential for the survival of melanocyte stem cells): Loss of hair pigmentation Knockouts of anti-apoptotic Bcl-2 family proteins *bcl-xL -/- mice (Motoyama et al., Science 267:1506-1510, 1995.) --- Animals die during embryogenesis --- Massive apoptosis in developing post-mitotic cells of the embryonic spinal cord, brain stem, and DRG --- Extensive apoptosis is also observed within the liver and hematopoietic systems --- Haploinsufficiency for Bcl-xL causes thrombocytopenia (Mason KD et al., Cell 128, 1173-1186, 2007). *mcl-1 -/- mice --- defects in survival and implantation of zygote (Rinkenberger et al., G&D 14:23, 2000) --- in conditional KO, critical in early hematopoiesis and B/T lymphopoiesis (Opferman JT et al., Nature 426, 671-676, 2003; Science 307, 1101-4, 2005) *bcl-w -/- mice --- defects in spermatogenesis (Print CG et al., PNAS 95, 12424-31, 1998; Ross AJ et al., Nat Genet. 18, 251-6, 1998)

12. Bcl-2 proteins control apoptosis BH3-only BCL-2 BH1-3 pro-apoptotic proteins adaptorcaspaseanti-apoptotic Bcl-2 proteins Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa

13. Bax and/or Bak knockouts *bak -/- mice (Lindsten et al., Mol. Cell 6:1389-1399, 2000) --- Animals develop normally --- bak -/- cells show normal apoptotic responses to both extrinsic and intrinsic apoptotic stimuli *bax -/- bak -/- mice: --- bak -/- bax -/- mice die perinatally with <10% animals reaching adulthood --- Adult bak -/- bax -/- mice display multiple phenotypic abnormalities (retain interdigital webs, imperforate vaginas, neurological abnormalities including deafness, and circling behavior, increased numbers of neuronal and hematopoietic progenitors; leading to elevated numbers of granulocytes and mature T/B lymphocytes and enlarged spleen and LN and infiltration of parenchymal organs; bak -/- bax -/- cells are resistant to death induced by neglect or irradiation but sensitive to Fas.) *bax -/- mice (Knudson et al., Science 270:96-99, 1995) --- Animals develop rather normally --- Increased numbers of sympathetic and motor neurons --- In vitro, bax -/- sympathetic neurons are resistant to cell death upon trophic factor withdrawal --- Male homozygous mice are sterile due to a paradoxical increase in the death of spermatogonia

14. *Unstimulated, Bax is monomeric and inactive in the cytosol, probably due to its concealed amino (or carboxy) termini. Bax is also normally “sequestered” in the cytosol as a result of binding to multiple proteins including 14-3-3 proteins, Ku70, the peptide humanin, HSP70, crystallins, and ARC or Apoptosis Repressor with Caspase recruitment domain). *Recently, Bax has been shown to be retro-translocated by Bcl-xL to block its proapoptotic effect (Edilich et al., Cell 145: 104-116, 2011). *Different from Bax, most Bak is constitutively expressed in the mitochondria by associating with VDAC2. *Bax and Bak modulate the UPR by a direct interaction with IRE1  (inositol-requiring enzyme 1  ) (Hetz et al., Science 312, 572, 2006) Bax and Bak play an important role in mitochondrial morphogenesis (Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ. Nature 443:658-62, 2006) *Bak has neuroprotective effect (Fannjiang Y et al., Dev. Cell. 4, 575-585, 2003). *In some cancer cells, Bax and Bak are mutated. Bax and Bak may have non-apoptotic and physiological functions

15. Bcl-2 proteins control apoptosis BH3-only BCL-2 BH1-3 pro-apoptotic proteins adaptorcaspaseanti-apoptotic Bcl-2 proteins Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa Chandra D, Liu J-W, Tang DG. Early mitochondrial activation and cytochrome c up-regulation during apoptosis. J. Biol. Chem. 277: 50842-50854, 2002.

16. Holo-cyto. c -COOH Apaf-1 CARD NOD WD-40 repeats (13) NH2- 1 1248 99-109 415 Shi, Y., Mol. Cell, 9, 459-470, 2002

17. Holo-cyto. c Apaf-1 -COOH CARD NOD WD-40 repeats (13) NH2- 1 1248 99-109 415 Apoptosome Formation can be Reconstituted using Recombinant Proteins --- A Process that Requires (d)ATP + +(d)ATP (0.1-0.5 mM)

18. Holo-cyto. c -COOH Apaf-1 CARD NOD WD-40 repeats (13) NH2- 1 1248 99-109 415 Physiological (mM) levels of (deoxy)N Caspase-9 activation Chandra D, Bratton SB, Person MD, Tian Y, Martin AG, Ayers M, Fearnhead HO, Gandhi V, and Tang DG. Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome c and inhibiting apoptosome. Cell 125: 1333-1346, 2006.

19. Holo-cyto. c -COOH Apaf-1 CARD NOD WD-40 repeats (13) NH2- 1 1248 99-109 415 Cytochrome c Apaf-1 Caspase-9 Caspase-3 Apoptosis Apoptosome Physiological (mM) levels of (deoxy)N

20. *Physiological levels of K + also inhibits cytochrome c-dependent apoptosome formation (JBC, 276, 41985-990, 2001). *Bao Q, Lu W, Rabinowitz JD, Shi Y. Calcium blocks formation of apoptosome by preventing nucleotide exchange in Apaf-1. Mol Cell. 2007 Jan 26;25(2):181- 92.

21. Caspases are executioners of apoptosis BH3-only BCL-2 BH1-3 pro-apoptotic proteins adaptorcaspaseanti-apoptotic Bcl-2 proteins Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa

22. ProdomainLarge subunitSmall subunit Linker p10 p20p3-26 Prodomains:In executioner caspases: ~3kd In initiator caspases: 10-26 kd Types of caspases: -Initiator caspases: caspase-2, -8, -9 and -10 -Executioner Caspases: caspase-3, -6 and -7 Caspase Structure: Caspase prodomains: D. Chandra

23. Mol. Cell 9, 459-470, 2002

24. Caspase substrates Proteosome components (Mol Cell 14, 81-93, 2004) Mitochondrial complex I p75 subunit (Cell 117, 773-786, 2004)

25. Bcl-2 proteins control apoptosis Bax Bak Apaf-1Caspase-9 Cyto c Caspase-3 Bad Noxa Death???

26. IAPs

27. Inhibitor-of-Apoptosis Proteins 604 BIR1BIR2BIR3RINGCARD BIR1BIR2BIR3RING CARD 618 BIR1 BIR2 BIR3 1403 BIR UBC 4845 BIR 142 1 BIR1BIR2BIR3 RING 497 XIAP cIAP-1 NAIP BRUCE Survivin cIAP-2 BIR3 RING 236 ILP-2 BIR RING 298 Livin S. Bratton

28. IAPs

29. Shi, Y. Mol. Cell, 9, 459-470, 2002 IAPs function as critical prosurvival molecules *IAPs (especially XIAP) are generally overexpreessed in multiple types of cancer cells Smac (second mitochondrial activator of caspases) apoptotic stimulation

30. Caspases may have apoptosis-independent/physiological functions Drosophila caspases (Cell 126, 583-596, 2006) --- Non-apoptotic caspase activity is required for proper sensory organ precursor development. Caspase-1 --- Promotes cell survival upon toxin challenge possibly through membrane repair (Cell 126, 1135-1145, 2006). --- Connects immunity (host defense) and apoptotic machinery (Bruey J-M., Cell 129, 45-56, 2007). Caspase-3 --- Caspase-3 activation is involved in cancer cell invasion (Cancer Res, 65, 9121-9125, 2005). Caspase-8 (Nature 419, 395, 2002; Genes & Dev 17, 883, 2003; Science 307, 1465, 2005) --- Targeted disruption of casp-8 in T-cell lineage leads to marked decrease in peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. --- Casp-8 ablation protected thymocytes and activated T-cells from CD95 ligation but not anti-CD3-induced apoptosis, or apoptosis activated by the mitochondrial stimuli --- Casp-8 mutant mice were unable to mount an immune response to viral infection --- Casp-8 required for NF-kB activation by antigen receptor --- Casp-8 promotes cell motility and calpain activity (Cancer Res., 66, 4273-4278, 2006) --- However, loss of caspase-8 potentiates neuroblastoma metastasis (Nature 439, 95-99, 2006) --- Caspase-8 interacts with androgen receptor (AR) and regulates AR-driven gene expression (Qi W et al., EMBO J. 26, 65-75, 2007)

31. Prosurvival mechanisms I. Endogenous prosurvival mechanisms II. Autophagy III. Induced prosurivival mechanisms

32. Autophagy *First recognized under EM early 1960s. *Also called macroautophagy: Self-eating to survive. *A unique form of membrane trafficking in which membrane compartments (autophagosomes) engulf both organelles and cytosolic macromolecules and deliver them to the lysosome for degradation.

33. Mizushima N. CDD, 12, 1535-1541, 2005

34. Autophagy *Molecular insights first came from studies in yeast, which expresses >20 autophagy-related genes (ATGs). *In yeast, two ubiquitin-like conjugation systems, Atg8-PE (phosphatidylethanolamine; Atg8 = LC3 in mammals) and Atg12-Atg5, are involved in forming the initial autophagic vesicles.

35. Yorimitsu and Klionsky. CDD, 12, 1542-1552, 2005 Autophagosome nucleation requires a complex containing Atg6 (Beclin 1 in mammals) that recruits the class III PI3K VPS34 to generate PI3P. Expansion of autophagosome membranes involves Atg12 and Atg8 (or LC3).

36. Autophagy: A Major Prosurvival Mechanism - In yeast, the primary function of autophagy is to maintain viability during starvation. - In mice, autophagy supports newborn survival at birth - in the absence of Atg5, mice do not survive neonatal starvation and die within a day after birth (Kuma et al., Nature 432, 1032, 2004). -In fact, autophagy-defective embryos fail to reach the blastocyct stage (Science 321:117, 2008). - In IL-3-dependent lymphocytes, cells without Bax and Bak survive for weeks due to autophagy - knockdown of Atg7 significantly reduces this survival (Lum et al., Cell 120, 237-48, 2005). - Autophagy-supported cell survival probably results from the products of self-degradation in lysosomes (e.g., amino acids), which support the TCA cycle to generate ATP. - Animal cells exhibit low levels of constitutive autophagy - a defense mechanism to eliminate aggregation-prone proteins (inclusion bodies) in, e.g., neurons and liver cells (absence of Atg5 and Atg7 in mouse brain induces neurodegenerative diseases (Hara T et al., Nature 441, 885, 2006; Komatsu M et al., Nature 441, 880, 2006). -Autophagy sequesters and kills the invading pathogens (Nakagawa I et al., Science 306, 1037, 2004). - Autophagy helps eliminate superfluous organelles (peroxisomes, mito., ER, etc) and maintain cellular homeostasis). - Defects in autophagy have been linked to liver diseases, neurodegeneration, Crohn’s disease, aging, metabolic syndrome and cancer *Indeed, autophagy functions as prosurvival mechanisms in cancer cells and mediate their resistance to therapeutics (Albedin MJ, et al., CDD, 14, 500-510, 2007; Katayama M et al., CDD, 14, 548-558, 2007; Amaravadi RK et al., JCI, 117:326-336, 2007).

37. Autophagy & Metabolism Rabinowitz JD & White E. Autophagy & Metabolism. Science 330: 1344-1348, 2010.

38. Autophagy & Metabolism Rabinowitz JD & White E. Autophagy & Metabolism. Science 330: 1344-1348, 2010.

39. Levine B and Yuan J. JCI, 115, 2679-2688, 2005 Yoshimori T et al., Cell 128, 833-836, 2007 Autophagy (Type II PCD) & Apoptosis (Type I PCD) *Autophagic cell death: nonapoptotic PCD (type II) *A large number of autophagosomes are present in dying cells: Cause-and-effect relationship unclear *The first example: mouse L929 fibroblasts treated with zVAD  cells undergo nonapoptotic cell death  knockdown of Atg7 or Beclin-1 reduced cell death + zVAD treatment somehow caused degradation of catalase resulting in increased ROS production followed by cell death (Yu L et al., Science 304, 1500-1502, 2004). *In Bax/Bak-DKO MEFs treated with chemicals such as etoposide cells die through ‘autophagy’ - cells do not die when Atg5 and Beclin-1 were silenced (Shimizu et al., NCB 6, 1221-1228, 2004). *’Autophagy’ might act upstream of apoptosis: apoptosis of CD4 T cells by HIV envelope glyco- proteins inhibited when Beclin-1 and Atg7 were silenced (Espert L et al., JCI, 116, 2161-72, 2006). *Problems: 1) Autophagy is a morphological and descriptive term; 2) The Atg proteins may have autophagy-unrelated functions; 3) Truncated Atg5 can bind Bcl-xL in mitochondria and causes apoptosis (Yousefi S et al., NCB 8, 1124-32, 2006). 4) Bcl-2 negatively regulates autophagy by binding to Beclin-1 (Pattingre S et al., Cell 122, 927-39, 2005). 5) ‘Autophagic cell death’ might be caused by excessive nonselective autophagy itself.

40. Yoshimori T et al., Cell 128, 833-836, 2007 Qu X et al., Cell 128, 931-46, 2007

41. Prosurvival mechanisms I. Endogenous prosurvival mechanisms II. Autophagy III. Induced prosurivival mechanisms Cell Apoptotic stimulation Survival Death

42. Two Apoptotic Pathways

43. Liu et al., ONCOGENE 2005 Induction of prosurvival molecules during apoptosis induction

44. Micheau O. & Tschopp J. Cell 114, 181-190, 2003 FasL (EMBO J. 23, 3175, 2004) --- FasL promotes tumor cell migration and invasion.

45. Barnhart & Peter, Cell 114, 148-150, 2003 Algeciras-Schimnich et al., PNAS 100, 11445, 2003 Barnhart et al., EMBO J. 23, 3175-3185, 2004

46. Prosurvival mechanisms I. Endogenous prosurvival mechanisms II. Autophagy III. Induced prosurivival mechanisms Cell Apoptotic stimulation Survival Death