Part I The p53-AKT network: A genetic network regulating cell survival and death The biological system studied in Part I (Chapters to 5) is a key genetic network regulating cell survival and death, which constitutes a connected network of cancerrelevant genes These genes interact among themselves through the various regulatory feedback loops inherent in the genetic network that generates rich and nonintuitive systems behaviors Therefore, a critical study of the interaction dynamics can lead to the understanding of how cells regulate the switch between cell survival and death in the presence of DNA damage and growth factor Chapter AKT versus p53 in a Network of Oncogenes and Tumor Suppressor Genes Regulating Cell Survival and Death The tumor suppressor protein p53 is often referred to as the ‘guardian of the genome’ because of its key role in inducing cells to die when, for example, their DNA is irreparably damaged This role is implemented by promoting the cell death program, called apoptosis, through mechanisms that can be both dependent and independent of p53’s transcriptional activity (Fridman and Lowe, 2003) At least half of known human cancers are associated with p53 gene mutations, and the majority of the remaining half involves malfunctions of the pathways regulating the protein’s activities (for reviews, see Lowe et al., 2004; Haupt et al., 2003; Oren, 2003; Vogelstein et al., 2000) In both mutated and wild type cases, p53 is prevented from causing apoptosis of cancer cells The serine/threonine kinase AKT (Protein Kinase B), on the other hand, promotes cell survival by inhibiting pro-apoptotic proteins (such as BAD and CASPASE-9) through phosphorylation (for reviews, see Franke et al., 2003; Nicholson and Anderson 2002; Datta et al., 1999) Thus, p53 and AKT influence the process of apoptosis in opposite ways Recent results summarized in the next section (Section 2.1) indicate that there are crosstalks between p53 and AKT involving gene transcription as well as post-translational protein modifications, which is characterized by a positive feedback loop between p53 and AKT This loop can also be described as a mutual antagonism between an oncoprotein, AKT, and a tumor suppressor protein, p53 (Harris and Levine, 2005; Gottlieb et al., 2002) This chapter summarizes the extensive biological literature pertaining to the interactions of genes in the p53-AKT network (Section 2.1), the regulation of the p53AKT network by growth factors and cellular stress (Sections 2.2 and 2.3) and finally, the regulation of caspase-dependent apoptosis (Section 2.4) by the p53-AKT network (Section 2.5) The regulatory networks reviewed here form the foundation of the mathematical models that are presented and analyzed in Chapters and 2.1 The p53-AKT network The complexity of p53 regulation is depicted in a recent review of Harris and Levine (2005), which focuses on the many positive and negative feedback loops in the regulatory networks Two of these loops are shown in a qualitative network of the p53-AKT network depicted in Figure 2-1 One is the important negative feedback loop between MDM2 and p53 (edges and 6) MDM2 binds and blocks the transactivation domain of p53, thereby inhibiting its transcriptional activity (Wu et al., 1993; Momand et al., 1992; Oliner et al., 1993) The p53-MDM2 complex then shuttles from the nucleus to the cytoplasm (Tao et al., 1999; Kubbutat et al., 1999) where MDM2 serves as an E3 ubiquitin ligase and targets p53 for ubiquitin-mediated proteosomal degradation (Wu et al., 1993; Haupt et al., 1997; Kubbutat et al., 1997) On the other hand, expression of the MDM2 gene is induced by p53 PTEN PIP3 p53 MDM2 AKT Apoptosis Figure 2-1 Qualitative network showing interactions involving p53 and AKT An arrow means a pathway that leads to activation or upregulation; a hammerhead represents inhibition or downregulation AKT is antagonized by p53 via edges to 3; and p53 is antagonized by AKT via edges and A p53-MDM2 negative feedback loop is shown by edges and A possible mutual activation loop between p53 and PTEN is shown by edges and PTEN: Phosphatase and Tensin homolog deleted on chromosome ten; PIP3: Phosphatidyl Inositol-3,4,5-trisphosphate; MDM2: Murine Double Minute Clone 2; AKT: Protein Kinase B The link between p53 and AKT (edges to 3) involves PIP3 and PTEN as shown in Figure 2-1 PIP3 is required for the recruitment of AKT to the plasma membrane where AKT gets phosphorylated and activated (Franke et al., 2003) One way by which p53 inhibits production of PIP3 indirectly is by inducing the expression of the lipid phosphatase PTEN (Stambolic et al., 2001; Harris and Levine, 2005) The chief biological function of PTEN is the dephosphorylation of PIP3, thereby inhibits its kinase activity (Gil et al., 2007); other phosphatase substrates of PTEN have remained rare (Trotman et al., 2003) Another way is by p53 repression of the catalytic subunit of PI3K, the enzyme that catalyzes the formation of PIP3 (Singh et al., 2002) Active AKT phosphorylates MDM2 (edge of Figure 2-1) causing the latter to translocate to the nucleus where it inhibits p53 (Zhou et al., 2001; Mayo and Donner, 2001) 10 As shown in Figure 2-1, a positive feedback loop (p53-AKT-MDM2-p53; edges to 5) and a negative loop (p53-MDM2-p53; edges and 6) are coupled via the MDM2-p53 interaction Mayo and Donner (2002) suggested an interesting interpretation of this coupling based on a report that the p53-induced transcriptional activation of MDM2 precedes that of PTEN (Stambolic et al., 2001) According to this interpretation, the p53-MDM2 negative feedback loop autoregulates the increase in p53 and delays p53-induced apoptosis to allow cells with DNA that are not irreversibly damaged or mutated to survive A subsequent p53-induced expression of PTEN triggers the p53-PTEN “amplification loop” which then suppresses the cell survival machinery; it is then suggested that this suppression is obligate for p53 apoptotic activity (Mayo and Donner, 2002) Recent studies showed that PTEN could activate p53 through phosphataseindependent mechanisms After association with P300, PTEN enhances acetylation of p53 at Lysine373 and Lysine382, and generates a subsequent PTEN binding site (Li et al., 2006) p53 that is bounded to PTEN-P300 has enhanced DNA-binding activity that augments transcription of PTEN and p21 (Li et al., 2006; Freeman et al., 2003; Luo et al., 2004) Because MDM2 cannot bind to this complex, it cannot mediate degradation of this specific form of p53 (Freeman et al., 2003; Zhou et al., 2003; Li et al., 2002) However, several biological questions still remain For instance, it is unclear whether PTEN-p53 complex could also enhance MDM2 transcription (an inherent p53 function) or whether it could still dephosphorylate PIP3 (an inherent PTEN function) So far, PTEN-p53 binding has been reported in U2OS (Li et al., 2006), Saos2 (Li et al., 2006; Freeman et al., 2003), ALL (Zhou et al., 2003) and MEF (Freeman et al., 2003) cells The relevance of this putative p53-PTEN positive 11 feedback loop (edges and of Figure 2-1) in the control of the cell survival-death switch will be addressed in Chapter (Section 5-11) Among the five proteins of the p53-AKT network, four of them are deregulated recurrently in numerous types of human tumors p53 is the most commonly mutated gene in leukemia, breast, gastric, ovarian and colorectal cancers (Royds and Iacopetta, 2006), followed by PTEN that is frequently mutated in glioblastoma, prostate, endometrial, breast and lung tumors (Depowski et al., 2001; Maier et al., 1998; Li et al., 1997; Steck et al., 1997) MDM2 and AKT, on the contrary, are often amplified and over-expressed (Altomare et al., 2005; Nicholson et al., 2002; Alarcon-Vargas et al., 2002) Thus, p53 and PTEN are major tumor suppressors (Mayo et al., 2002; Mayo and Donner, 2002; Cantley et al., 1999) whereas MDM2 and AKT are major oncogenes (Alarcon-Vargas et al., 2002; Momand et al., 2000) 2.1.1 Cell types that activate the p53-AKT genetic network As expected from the mutual inhibition between p53 and AKT, inverse correlation between protein levels of p53 and phosphorylated (active) AKT have been reported recently in numerous cell lines, such as human breast epithelial cancer (MCF-7 and HBEC), primary human embryonic kidney (HEK 293), human sarcoma osteogenic (Saos-2), human colon tumor (EB1), primary human keratinocytes and foreskin fibroblasts, mouse fibroblasts (MEF and NIH3T3), mouse lymphoid and hematopoietic (DA-1 and BaF3), mouse epidermal (CI41) and mouse hippocampal 12 neurons (Gottlieb et al., 2002; Su et al., 2003; Wang et al., 2005; Ogawara et al., 2002; Zhou et al., 2001; Singh et al., 2002; Yamaguchi et al., 2001; Mayo and Donner, 2001) However, because experimentalists have not attempted a systems analysis of the p53-AKT genetic network, the specific cell types in which the p53AKT network is active are extracted from separate publications Both the p53-MDM2 feedback loop and the PI3K/PIP3/AKT pathway are very well-studied during the last decade The p53-MDM2 loop is active in practically all cell types in response to various cellular stress (Vogelstein et al., 2000; Momand et al., 2000; Bond et al., 2005; Piette et al., 1997; Momand and Zambetti, 1997; JuvenGershon and Oren, 1999; Moll and Petrenko, 2003; Iwakuma and Lozano, 2003; Alarcon-Vargas and Ronai, 2002; Horn and Vousden, 2007) while the PI3K/PIP3/AKT pathway is a general survival pathway that is activated by diverse survival and growth factors whose activity is essential for cell survival in most cell types (Nicholson and Anderson, 2002; Kandel and Hay, 1999; Franke et al., 2003; Coffer and Woodgett, 1991; Jones et al., 1999; Bellacosa et al., 1993; Brazil and Hemmings, 2001; Datta et al., 1999; Kauffmann-Zeh et al., 1997; Blair et al., 1999; Chen et al., 1998; Crowder and Freeman, 1998, 1999; Eves et al., 1998; Gerber et al., 1998; Häusler et al., 1998; Kennedy et al., 1997; Khwaja and Downward, 1997; Khwaja et al., 1997; Kulik et al., 1997; Kulik and Weber, 1998; Philpott et al., 1997; Rohn et al., 1998; Songyang et al., 1997; Xiong and Parsons, 1997; Liu et al., 1999; Gold et al., 1999; del Peso et al., 1997; Ahmed et al., 1997; Parry et al., 1997; BlumeJensen et al., 1998; Gibson et al., 1999; Leverrier et al., 1999; Kontos et al., 1998; Gautreau et al., 1999) Similarly, PTEN protein expression is constitutive in all tissues and essential at all times (Salmena et al., 2008; Li et al., 1998; Chung et al., 13 2005; Perren et al., 1999; Di Cristofano et al., 1998; Podsypanina et al., 1999; Stambolic et al., 1998, 2000, 2001; Suzuki et al., 1998; Li and Sun, 1998; HaasKogan et al., 1998; Shan et al., 2000; Wu et al., 1998; Sun et al., 1999), whose key biological function is to inhibit the PI3K/PIP3/AKT-mediated survival pathway by deactivating PIP3 through dephosphorylation (Weng et al., 1999; Li et al., 1998; Li and Sun, 1998; Wang et al., 2000; Shan et al., 2000; Haas-Kogan et al., 1998; Ramaswamy et al., 1999; Zhou et al., 2003; Su et al., 2003; Wu et al., 1998; Stambolic et al., 1998, 2001; Dahia et al., 1999; Maehama and Dixon, 1998) On the other hand, the remaining interactions in the p53-AKT network namely, p53-PTEN and AKT-MDM2, are elucidated recently p53-dependent transcription of PTEN (p53-PTEN) has thus far been detected in human breast cancer epithelial (MCF-7), human colon carcinoma (HCT116 and EB1), human sarcoma osteogenic (Saos-2), human adenocarcinoma (H460), human lung fibroblast (WI38), human osteosarcoma (U2OS), human epithelial carcinoma (HeLa), human hepatocellular carcinoma (HepG2), human glioblastoma (A172 and U87MG), human prostate carcinoma (PC3), human acute myeloid leukemia (AML-5), primary human bronchial epithelial (HBEC), mouse embryonic fibroblasts (MEF), mouse erythroleukemia (DP16) as well as in mouse skeletal muscle, heart, white fat, liver, kidney, lung, small intestine, colon, skin and brain cortex (Feng et al., 2007; Wang et al., 2005; Stambolic et al., 2001; Trotman and Pandolfi, 2003; Singh et al., 2002; Tang and Eng, 2006a, 2006b) AKT phosphorylation of MDM2 (AKT-MDM2) has been observed in human breast epithelial and tissues (MCF-7, MDA-468, T47D), human non-small lung carcinoma (H1299), human osteosarcoma (U2OS), human sarcoma osteogenic (Saos-2), human fibroblasts (MRC-5), human embryonic kidney 14 (293T and HEK293), human primary keratinocytes cells, mouse fibroblasts (NIH3T3 and MEF) (Gottlieb et al., 2002; Mayo et al., 2002; Mayo and Donner, 2001; Ashcroft et al., 2002; Ogawara et al., 2002; Zhou et al., 2001) In summary, cell types in which every interaction in the p53-AKT network is active are observed at least in human breast cancer epithelial (MCF-7), human sarcoma osteogenic (Saos-2), human osteosarcoma (U2OS), as well as in human and mouse fibroblasts This list of cell types is incomplete because firstly, it is impossible to review the entire literature pertaining to each part list of the p53-AKT network and secondly, current active research activities particularly in the p53-PTEN and AKTMDM2 interactions will expand the cell types used in experiments As such, the list of cell types described here is clearly an underestimation 2.2 Regulating the p53-AKT network The p53-AKT network is regulated by signals emanating from growth factors and cellular stress (e.g DNA damage), which results in either cell survival or death, as described below 2.2.1 Growth-factor stimulation Growth factors promote cell survival via the PI3K/AKT network, one of the central signal transduction pathways that comprises of PI3K, PDK1, PIP3, AKT and PTEN 15 (Lian and Cristofano, 2005), as depicted in Figure 2-2 PI3K is a heterodimer composing of two subunits namely, p85 regulatory unit and p110 lipid catalytic unit Upon binding of growth factors such as IGF, EGF, FGF, and NGF etc to their respective cell receptors, p85 subunit of PI3K is recruited to the receptors at the cell membrane, where the activated p110 catalytic subunit phosphorylates PIP2 to form PIP3 (Deleris et al., 2006; Viniegra et al., 2005; Datta et al., 1999; Jarpe et al., 1998) Together with PDK1, PIP3 phosphorylates AKT at Threonine308 (Franke et al., 2003; Scheid et al., 2002); and Serine473 by a yet to be identified kinase (Deleris et al., 2006); both resides must be phosphorylated to fully activate AKT kinase activity Active AKT thereby promotes cell survival through phosphorylation that activates pro-survival substrates such as oncoprotein MDM2 while deactivates pro-apoptotic substrates (Ahmed et al., 1997; Kennedy et al., 1997; Songyang et al., 1997; Khwaja et al., 1997; Dudek et al., 1997; Kulik et al., 1997) Growth factor-bound receptors PI3K PIP2 PIP3 PDK1 AKT AKTa Cell survival PTEN Figure 2-2 The PI3K/AKT network Growth factors promote cell survival via the PI3K/AKT network (see main text for details) AKTa denotes active AKT protein PI3K: Phosphatidylinositol 3-Kinase; PIP2: Phosphatidyl 16 description of the DNA damage signal transduction pathway) In the case of p53, these modifications lead to nucleus translocation, tetramerization, diminished binding affinity to MDM2 and activation of transcriptional activity (Stommel et al., 1999; Chene, 2001; Shieh et al., 1997) As a result, the level of transcriptionally active p53 is upregulated in the nucleus where it induces expression of PTEN (to inhibit the PI3K/AKT survival pathway) as well as specific genes that impinges on cell cycle arrest, DNA repair and/or apoptosis (Levine, 1997) Modified MDM2, on the other hand, has attenuated binding affinity to p53, which impedes its ability to ubiquitinate p53 Unexpectedly, under this circumstance, MDM2 ubiquitinates itself, resulting in a short half-life (Stommel and Wahl, 2004) These post-translational modifications on the p53-MDM2 negative feedback loop have been cited as a reason for the observed oscillations in p53 and MDM2 protein levels (Lev Bar-Or et al., 2000; Lahav et al., 2004) in human breast cancer epithelial MCF-7 cells; IR induced DNA damage also results in p53-dependent upregulation of PTEN mRNA in MCF-7 cells (Tang and Eng, 2006a; Tang and Eng, 2006b) The biological implications of p53 oscillations are studied in detail in Chapter 2.3 DNA damage signal transduction pathways Upon exposure to IR, the major cellular events occurs generally in the following sequence: formation and detection of DSBs, transmission of DNA damage signal by transduction pathways and finally, cellular outcomes such as cell cycle arrest, damage repair and apoptosis executed by downstream effectors (Rouse and Jackson, 2002; Norbury and Zhivotovsky, 2004; Zhou and Elledge, 2000) The biochemical and 18 biophysical mechanisms of these events are however far from being completely elucidated (McGowan and Russell, 2004; Durocher and Jackson, 2001; Zhou and Elledge, 2000) Despite that, existing notion of the DNA damage signal transduction pathways regulating post-translation modifications of p53 and MDM2, which is of most relevance in this study, is complex (Figure 2-3) In general, the part lists of the damage signal transduction pathway can be categorized into primary and secondary transducers MDM2 ATM PP2A 53BP1 BRCA1 DNA-PK ATR CHK2 WIP1 p53 Figure 2-3 DNA damage signal transduction pathways regulating p53 and MDM2 Signal transduction pathways regulating the activation (arrows) and suppression (hammerheads) of p53 and MDM2, following IR-induced formation of DSBs The primary signal transducers are ATM, ATR and DNA-PK However, crosstalking among the secondary signal transducers are prevalent, as indicated by the numerous network edges ATM, ATR and DNA-PK kinases are primary transducers, which belong to the phosphatidylinositol kinase-related kinase family ATM is present as inactive dimers in undamaged cells but is rapidly phosphorylated and thereby dissociates into active monomers in the presence of DSBs (Bakkenist and Kastan, 2003) Active ATM phosphorylates p53 (Saito et al., 2002; Kapoor et al., 2000), MDM2 (Khosravi et al., 1999; Stommel and Wahl, 2004), CHK2 (Matsuoka et al., 1998; Ahn et al., 19 2000), BRCA1 (Cortez et al., 1999), 53BP1 (Fernandez-Capetillo et al., 2000) and maybe AKT (Fayard et al., 2006; Viniegra et al., 2005) p53 is stabilized and activated after being phosphorylated at multiple serine residues (Saito et al., 2002) at namely, 9, 15 (inhibits binding to MDM2), 20 and 46 (important for apoptotic activity) On the other hand, ATM-mediated phosphorylation of MDM2 augments MDM2 auto-ubiquitination (Stommel and Wahl, 2004; Khosravi et al., 1999) Although ATM is a key signal transducer in DNA damage response, alternate pathways exist as ATM-null cells still show phosphorylations of p53, MDM2, BRCA1 and CHK2 albeit with delayed kinetics after IR (Zhou and Elledge, 2000) For instance, ATR has been implicated in the late phosphorylation of p53 at Serine15 after IR (Tibbetts et al., 1999; Lakin et al., 1999; Hall-Jackson et al., 1999) Moreover, DNA-PK could bind to DSBs (Zhou and Elledge, 2000; Durocher and Jackson, 2001) after which it phosphorylates MDM2 at Serine17 (negates binding to p53) (Stommel and Wahl, 2004; Mayo et al., 1997) and p53 at serine residues at 15, 37, 46 and 392 (Kapoor et al., 2000; Komiyama et al., 2004; Jack et al., 2004; Shieh et al., 1997) Indeed, DNA-PK deficient cells display delayed and attenuated p53dependent activation of p21 and MDM2 proteins (Kachnic et al., 1999) Secondary signal transducers are direct downstream substrates of primary transducers that include CHK2, 53BP1 and BRCA1 (Figure 2-3) In particular, CHK2 is a common target of ATM and DNA-PK (Li and Stern, 2005) CHK2 exists as inactive monomers in unperturbed cells (Ahn et al., 2004) but undergoes multiple intermolecular phosphorylations especially at Threonine68 following DSBs formation (Lee and Chung, 2001; Schwarz et al., 2003), after which it dimerizes and becomes fully activated (Schwarz et al., 2003; Wu and Chen, 2003) Active CHK2 20 phosphorylates p53 at Serine20 and thereby blocks p53-MDM2 interaction (Caspari, 2000; Hirao et al., 2000; Chehab et al., 2000; Shieh et al., 2000; Takai et al., 2002); moreover, by cooperating with DNA-PK, CHK2 could also phosphorylates p53 at Serine15 (Kapoor et al., 2000; Jack et al., 2004) Similarly, BRCA1 phosphorylates p53 efficiently at Serine15 after activation by ATM (Foray et al., 2003; Fabbro et al., 2004) 53BP1, on the other hand, is required for p53 accumulation in response to IR (Wang et al., 2002) Remarkably, crosstalks among the secondary signal transducers exist BRCA1 is phosphorylated by CHK2 (Lee et al., 2000) while 53BP1 played a partially redundant role in phosphorylating BRCA1 and CHK2 (Wang et al., 2002) Opposing the effects of kinases are de-phosphorylating enzymes called phosphatases such as PP2A and WIP1 that extinguish the DNA damage signals by deactivating CHK2 and p53 (Figure 2-3) PP2A dephosphorylates CHK2 at Threonine68 (Liang et al., 2006), which prevents currently inactive CHK2 from being activated, but has no effect on currently active CHK2 In addition, active CHK2 is dephosphorylated by WIP1 at Threonine68, Serine516 and Serine33 (Oliva-Trastoy et al., 2007) Interestingly, WIP1 is involved in a negative feedback loop with p53, i.e., p53 activates WIP1 transcription (Fiscella et al., 1997) whereas WIP1 dephosphorylates p53 at Serine15 (Lu et al., 2005) Clearly, there are redundancies in the DNA damage signal transduction pathways that underscore the importance of rapid and appropriate response to DNA damage; accurate transmission of genetic information to daughter cells is a basic requirement for the survival of multi-cellular organisms (Zhou and Elledge, 2000) Nevertheless, not all of the pathways may be simultaneously active and their relative 21 contributions in the post-translational modifications of p53 and MDM2 proteins could depend on cell lines and extent of DNA damage 2.4 Programmed cell death There are broadly two types of cell deaths – programmed cell death (PCD) and necrosis (Fiers et al., 1999) Unlike necrosis, PCD is mediated by specialized and complex biochemical pathways that degrade remnants of a death cell cleanly, and thereby prevents unnecessary immune response PCD or apoptosis is thus the default mode of removing unwanted cells PCD is further classified into caspase-dependent and caspase-independent apoptosis (Lawen, 2003; Hengartner, 2000) In the former, a family of proteases termed executioner caspases (cysteine-containing aspartatespecific proteases) executes apoptosis by cleaving numerous specific cellular substrates at aspartate residue sites (Kumar, 2007; Timmer and Salvesen, 2007; Nicholson, 1999) Dormant in surviving cells, caspases must be processed (by irreversible removal of a peptide sequence from the protein) to activate their apoptotic activities However, once intracellular threshold quantities of caspases are activated, a point of no return is reached whereby a cell commits irrevocable to death Caspaseindependent apoptosis (Borner and Monney, 1999; Kitanaka and Kuchino, 1999), on the other hand, is comparatively less well studied and prevalent partly because it is induced in circumstances under which there is insufficient cellular resource (such as ATP) to activate the default caspase-mediated apoptotic pathways This section gives an overview of the caspase-dependent pathways since they are impinged extensively by both p53 and AKT 22 2.4.1 Caspase-dependent apoptotic pathways As depicted in Figure 2-4, caspases are activated via the extrinsic and intrinsic apoptotic pathways These pathways are activated by myriad stimulations such as UV or gamma radiation, chemotherapeutic drugs, growth factor withdrawal, and upregulation of oncogenes (Jin and El-Deiry, 2005) The extrinsic pathway (see reviews: Jin and El-Deiry, 2005; Lawen, 2003) is initiated through death receptors such as FAS (APO-1/CD95), TNF, DR3 (TRAMP), DR4 (TRAIL-R1), and DR5 (TRAIL-R2) Among them, part lists of FAS-mediated pathway are well characterized Upon binding of its native ligand (FASL), FAS selfoligomerizes which then recruits FADD adaptor proteins to its cytoplasmic domains, forming DISC (Death Inducing Signaling Complex) The main function of DISC is to recruit multiple PROCASPASE-8 molecules to close proximity so as to induce mutual cleaving of their protein peptides (Muzio et al., 1998) This processing step produces one of the initiator caspases, CASPASE-8 DISC could activate another initiator caspase, CASPASE-10, by this similar mechanism Remarkably, a positive feedback loop exists between these two initiator caspases in which they undergo mutual activation, thus amplifying the death signal (Sprick et al., 2002; Wang et al., 2001) Subsequently, CASPASE-8 cleaves PROCASPASE-3 and PROCASPASE-7 to form two of the respective executioner caspases, CASPASE-3 and CASPASE-7 while CASPASE-10 cleaves PROCASPASE-3 only 23 INTRINSIC EXTRINSIC ligands mitochondria receptors cell membrane DISC CYTOCHROME C tBID FADD APAF-1 APOPTOSOME BID PROCASPASE-9 PROCASPASE-8, -10 CASPASE-8, -10 CASPASE-9 PROCASPASE-3, -6, -7 CASPASE-3, -6, -7 Cell Death Figure 2-4 Major steps of the intrinsic and extrinsic caspase-dependent apoptotic pathways The main function of the intrinsic (left panel) and extrinsic (right panel) pathways is to activate the executioner caspases, CASPASE-3, -6 and -7, the key proteins to carry out the cell death processes (the figure is adapted from Zheng and Flavell, 2000 with permission from Nature Publishing Group) Interestingly, both pathways share similar network structure, which involves the formation of a complex for recruiting and activating initiator caspases (CASPASE-8, -9 and -10) Subsequently, initiator caspases of the two pathways activate the executioner caspases Interestingly, the extrinsic pathway impinges on the intrinsic pathway through BID (see text for details) The intrinsic pathway (see reviews: Jin and El-Deiry, 2005; Lawen, 2003), also called the mitochondria pathway, requires the release of CYTOCHROME C from the mitochondria to the cytoplasm (Garrido et al., 2006) where it forms a high molecular weight complex called the APOPTOSOME with APAF-1 and ATP APOPTOSOME mainly recruits and activates PROCASPASE-9 to produce another 24 initiator caspase, CASPASE-9 (Bao and Shi, 2007) Similarly, CASPASE-9 activates several executioner caspases, including CASPASE-3 and CASPASE-7, which converges with the extrinsic pathway Interestingly, the extrinsic pathway activates the intrinsic pathway via the apoptotic BID protein (Wang et al., 1996; Gross et al., 1999b) CASPASE-8 of the extrinsic pathway cleaves BID to form tBID, which then translocates to the mitochondria membrane to promote the release of CYTOCHROME C (Li et al., 1998; Grinberg et al., 2002) Because apoptosis leads to death, its tight regulation is critical Indeed, to prevent untimely cell death, two general mechanisms to quench the apoptotic signals are inherent in the pathways, as described in the following paragraphs The first mechanism inhibits the activation of caspases In the extrinsic pathway, c-FLIP competes with PROCASPASE-8 to bind to DISC and prevents the processing of PROCASPASE-8 (edges and of Figure 2-5) (Irmler et al., 1997) In the intrinsic pathway, the BCL2 protein family is the chief regulator of the release of CYTOCHROME C from the mitochondria (Cory and Adams, 2002; Gross et al., 1999a) The anti-apoptotic members, BCL-2 and BCL-XL, protect the mitochondria membrane potential and thus inhibit the release of CYTOCHROME C (edge 3) (Schwartz and Hockenbery, 2006) They are however antagonized through direct binding of their pro-apoptotic counterparts (edges and 5; BAD, BAX, BID, BIM, BAK, NOXA and PUMA) (Fletcher and Huang, 2006) The relative abundance of the anti-apoptotic and pro-apoptotic members has been proposed as the tipping point between cell survival and death (Walensky, 2006) 25 FASL DISC c-FLIPS BAD BAX BIM BAK NOXA PUMA c-FLIPL 13 C10 C8 14 19 24 16 23 15 tBID 18 C3 IAPs C7 BCL-2 17 25 BCL-XL C6 20 21 CYTOCHROME C IAPs C9 C2 22 DIABLO OMI 12 SURVIVIN 10 APOPTOSOME 11 IAPs Figure 2-5 Regulatory points of the caspase-dependent apoptotic pathways Proteins that are involved in the quenching of the apoptotic signal are indicated in gray Arrowheads denote activation whereas hammerheads denote inhibition The extrinsic pathway is shown in yellow background the intrinsic pathway is shown in blue background Additionally, the regulatory points of p53 and AKT proteins are indicated respectively in red and blue edges (see Section 2.4.2) Note: the caspases are abbreviated as “C”, e.g., C3 denotes CASPASE-3 The second quenching mechanism degrades activated caspases Activated caspases are antagonized by various members of the anti-apoptotic IAP family such as XIAP, c-IAP, IAP-1 and IAP-2, in which they bind to caspases such as CASPASE-3 (edges and 7), CASPASE-7 (edge 8) and CASPASE-9 (edge 9) and target them for ubiquitin-mediated degradation (Deveraux and Reed, 1999; Salvesen and Duckett, 2002) The anti-apoptotic activities of IAPs are in turned antagonized by pro26 apoptotic DIABLO (SMAC) and OMI (HTRA2) proteins that are released from the mitochondria (Verhagen et al., 2000; Liu et al., 2000; Wu et al., 2000; Hu et al., 2003; Suzuki et al., 2001; Yang et al., 2003; van Loo et al., 2002) Notably, binding of IAPs to DIABLO/OMI inhibits both of their biochemical activities, thus forming a mutual antagonism feedback loop (edges 10 and 11) In addition, pro-apoptotic DIABLO and OMI proteins are in turned antagonized by anti-apoptotic SURVIVIN protein (edge 12) (Ambrosini et al., 1997; Li et al., 1998) Therefore, these antagonistic feedback loops negate erroneous activation of caspase cascades caused by random spike in the apoptotic signal Conversely, to commit irrevocably to cell death, several mechanisms for amplifying the apoptotic signals exist Of utmost significance is the caspase activation cascade, which is characterized by mutual activations among executioner and initiator caspases (Budihardjo et al., 1999; Earnshaw et al., 1999; Shi, 2002) These positive feedback loops result in rapid upregulation of executioner caspases; at least five such loops are elucidated (edges 13 and 14; edges 15 and 16; edges 15, 17 and 18; edges 19, 16 and 14; edges 20 and 21) Moreover, active caspases further upregulate the activation of more caspases by inducing the release of CYTOCHROME C through the formation of tBID and inhibition of anti-apoptotic BCL-2 protein (edges 22 to 25) In summary, the perplexing interactions among disparate anti-apoptotic and pro-apoptotic proteins underscore the importance of timely regulation of cell survival and death in the response to myriad survival and/or death signals 27 2.5 Regulation of apoptosis by the p53-AKT network Remarkably, as depicted in Figure 2-5, p53 (red edges) and AKT (blue edges) impinge extensively on the extrinsic and intrinsic apoptotic pathways; the regulatory targets of p53 and AKT are summarized in Figure 2-6 p53 induces apoptosis primarily by expressing pro-apoptotic gene targets such as CASPASE-6 (MacLachlan and El-Deiry, 2002), FAS (Owen-Schaub et al., 1995; Sheard et al., 1997; Lin et al., 2002), APAF-1 (Fortin et al., 2001; Rozenfeld-Granot et al., 2002; Ho et al., 2003), DR5 (Takimoto and El-Deiry, 2000) and pro-apoptotic members of the BCL2 family encompassing BAX (Miyashita and Reed, 1995), BID (Sax et al., 2002), BAD (Jiang et al., 2006), BAK (Pohl et al., 1999), NOXA (Oda et al., 2000; Seo et al., 2003) and PUMA (Nakano and Vousden, 2001); PUMA could stabilize p53, which completes a positive p53-PUMA feedback loop (Chipuk et al., 2005) In addition, p53 could also promote apoptosis through transcription-independent mechanisms For instance, expression of pro-survival BCL-2 gene is repressed by p53 (Hemann and Lowe, 2006; Miyashita et al., 1994) When localized in the cytoplasm or mitochondria membrane, p53 binds and inhibits pro-survival BCL-2 and BCL-XL proteins (Mihara et al., 2003), while on the other hand, binds and activates pro-apoptotic BAK (Leu et al., 2004) and BAX (Chipuk et al., 2004) proteins 28 PUMA p53 A1 BCL-XL IAPs XIAP c-FLIP NF-κB CREB AKT BCL-2 BCL-2 BCL-XL APAF-1 BAK BAX BID CASPASE-6 DR5 FAS NOXA BAD CASPASE-9 FOXO OMI BCL-6 BIM FASL Figure 2-6 Regulatory targets of p53 and AKT p53 and AKT targets of the caspase-dependent apoptotic pathway are shown Pro-apoptotic targets are shown in red while pro-survival targets are shown in blue Arrowheads denote activation or upregulation whereas hammerheads denote inhibition or downregulation AKT promotes cell survival primarily by phosphorylating pro-apoptotic and pro-survival substrates For the former, AKT-mediated phosphorylation inhibits proapoptotic proteins such as CASPASE-9 (Cardone et al., 1998; Fujita et al., 1999), OMI (Yang et al., 2007), BAD (del Peso et al., 1997; Datta et al., 1997) and FOXO (Brunet et al., 1999; Kops et al., 1999) Phosphorylated CASPASE-9 and OMI cannot bind to their substrates for cleaving whereas phosphorylated BAD cannot bind and antagonizes pro-survival BCL-XL protein, and is subsequently sequestered by 143-3 chaperone protein (Datta et al., 2000) Phosphorylated FOXO is unable to express pro-apoptotic genes such as FASL, BIM and BCL-6 after DNA damage (van der Horst and Burgering, 2007), and is exported out of the nucleus for degradation (Biggs et al., 1999) In contrast, AKT-mediated phosphorylation activates prosurvival proteins such as XIAP (Dan et al., 2004), NF-κB (Ozes et al., 1999; Romashkova and Makarov, 1999) and CREB (Du et al., 1998) Phosphorylated XIAP has reduced susceptibility to ubiquitin-mediated degradation while phosphorylated NF-κB and CREB can express pro-survival genes – NF-κB expresses BCL-XL (Chen 29 et al., 2000), A1 (BFL-1, a pro-survival member of the BCL2 family) (Zong et al., 1999) and several IAPs (Chu et al., 1997) and CREB expresses BCL-2 (Riccio et al., 1999) Lastly, through unknown mechanisms, AKT upregulates c-FLIP (Uriarte et al., 2005; Skurk et al., 2004; Plate, 2004; Suhara et al., 2001), which inhibits the activation of initiator caspase of the extrinsic pathway 2.6 Review of apoptotic mathematical models Various aspects of the caspase-dependent apoptotic pathways have been described in mathematical models A common goal in these models is to identify and study the origin of the apoptotic thresholds Elucidating the biochemical or biophysical mechanisms governing the apoptotic threshold is an important biological problem as experimental observations suggest the presence of apoptotic thresholds In particular, induction of cell death does not follow a graded response, in other words, a minimum threshold amount of apoptotic stimulus is required to induce apoptosis Generally, the models analyze specific sub-networks of the entire apoptosis pathway and they can be grouped into two distinct classes based on their key predictions The first class of models predicts the existence of bistability (two steady states) between cell-survival and cell-death steady states as the origin of a cellular apoptotic threshold Possible causes of bistability have been proposed: positive feedback loops between CASPASE-3 and CASPASE-9 (edges 20 and 21 in Figure 25) (Legewie et al., 2006), CASPASE-3 and CASPASE-8 (edges 15 and 16 in Figure 2-5) (Eissing et al., 2004, 2005), and a hypothesized kinetic cooperativity in the 30 formation of the APOPTOSOME complex (see Figure 2-4) (Bagci et al., 2006) On the other hand, in the second class of models, the initial amount of pro-survival proteins is predicted to set the apoptotic threshold (Stucki and Simon, 2005; Bentele et al., 2004; Hua et al., 2005, Aldridge et al., 2006; Fussenegger et al., 2000) For instance, quenching of IAPs is a minimum threshold to overcome to induce cell death (Stucki and Simon, 2005) In addition, modeling and experimental studies show that the quantity of c-FLIP, an inhibitor of DISC, sets the main apoptotic threshold in Type I cells (apoptosis occurs predominantly via the extrinsic or DISC pathway, Figure 2-4) (Bentele et al., 2004) whereas the balance of BCL2 protein family among its pro-survival and pro-apoptotic members sets the apoptotic threshold in Type II cells (apoptosis occurs predominantly via the intrinsic or APOPTOSOME pathway, Figure 2-4) (Hua et al., 2005) Nevertheless, these models not study known regulatory pathways upstream of the apoptotic pathways that could impinge on the apoptotic thresholds Despite the many regulatory points at which p53 and AKT act upon the apoptotic pathway (Figures 2-5 and 2-6), it is somewhat surprising that no modeling study has been performed to analyze the p53-AKT network Therefore, Part I of this thesis aims to analyze this particular network in detail Interestingly, the p53-AKT models also predict the existence of bistability This thesis however, does not focus on the detailed analysis of the apoptotic pathways 31 2.7 Summary The multiple nodes in the apoptotic pathways on which p53 and AKT impinge underscore the importance of the p53-AKT network in the control of cell survival and death The biological importance of the p53-AKT network is further emphasized by the fact that it constitutes a connected network of cancer-relevant genes Taken together, these features underline the significance of studying the p53-AKT network 32 ... skin and brain cortex (Feng et al., 20 07; Wang et al., 20 05; Stambolic et al., 20 01; Trotman and Pandolfi, 20 03; Singh et al., 20 02; Tang and Eng, 20 06a, 20 06b) AKT phosphorylation of MDM2 (AKT-MDM2)... (CI41) and mouse hippocampal 12 neurons (Gottlieb et al., 20 02; Su et al., 20 03; Wang et al., 20 05; Ogawara et al., 20 02; Zhou et al., 20 01; Singh et al., 20 02; Yamaguchi et al., 20 01; Mayo and. .. kidney 14 (29 3T and HEK293), human primary keratinocytes cells, mouse fibroblasts (NIH3T3 and MEF) (Gottlieb et al., 20 02; Mayo et al., 20 02; Mayo and Donner, 20 01; Ashcroft et al., 20 02; Ogawara