Apoptosis And Inhibitor of apoptosis proteins Family: A
Background
Apoptosis is an orchestrated biological cellular process that occurs in physiological and pathological conditions(1). It is essential for regulating development, homeostasis, and immune-system function in organisms(2). In mammalian cells, apoptosis is mediated by a family of cysteine proteases named caspases which are initially expressed in cells as inactive procaspase precursors and are activated by two pathways, the extrinsic ( or death receptor) and intrinsic (or mitochondrial) apoptotic pathways(1).
The extrinsic pathway is activated by the binding of ligands such as Fas ligand (FasL) and tumour necrosis factor (TNF) to death receptors on the cell surface, FAS and the TNF receptor (TNFR), respectively, which leads to the formation of the death-induced signalling complex (DISC)(3)(4). DISC recruits caspase-8 and promotes the cascade of procaspase activation that follows(5).
The intrinsic pathway is triggered by extracellular and intracellular stresses, such as high cytosolic [ca+2 ], hypoxia, severe oxidative stress, DNA damage(5), which results in the permeabilization of the outer mitochondrial membrane, the release of pro-apoptotic molecules such as cytochrome C and others into the cytoplasm(6), the formation of the apoptosome- a large protein complex that is made up of cytochrome C, apoptotic protease activating factor 1 (APAF1) and caspase-9 – and caspase activation(7).
On the other hand, cell death is also modified by other mitochondrial proteins such as apoptosis-inducing factor(AIF), second mitochondria- derived activator of caspase (Smac), direct IAP Binding protein with low PI (DIABLO) and Omi/high temperature requirement protein A (Htr A2)(7). Smac/ DIABLO or Omi/HtrA2 induces cell death independently of caspase activation by counteracting inhibitor of apoptosis (IAP)- mediated caspase inhibition(7)(8)( Fig. 1).
The upstream caspase for the intrinsic pathway is caspase 9, while that of the extrinsic pathway is caspase 8. The intrinsic and extrinsic pathways cleave the precursor forms of effector caspases, such ascaspase-3, caspase-6 and caspase-7(9). Activated effector caspases cleave many vital cellular proteins such as protein kinases, cytoskeletal proteins, DNA repair proteins and inhibitory subunits of endonucleases family and break up the nuclear scaffold and cytoskeleton(9). They also activate DNAase, that further degrade nuclear DNA(10), which together contribute to the typical morphological changes in apoptosis.
Dysregulation of apoptosis has been implicated in numerous pathological conditions, including cancer(1). Besides, targeting the apoptotic pathways for cancer treatment is supported by several findings emphasizing the role of aberrant apoptosis in tumorigenesis and also resistance to anticancer treatment. Evasion from apoptosis is critical for tumor growth and a hallmark of cancer(11). One of the mechanisms by which evasion of apoptosis occurs is disrupted balance of pro-apoptotic and anti-apoptotic proteins(1). A delicate balance between pro-apoptotic and anti-apoptotic mechanisms determines whether a cell death signal can activate the apoptotic program. It is not the absolute quantity but rather the ratio of these pro-and anti-apoptotic proteins that controls the regulation of cell death. In this balance, pro-apoptotic proteins activate apoptosis and anti-apoptotic proteins inhibit apoptosis(12)(13). Inhibitors of apoptosis protein (IAPs) are important members of the anti-apoptotic family of proteins that can inhibit caspase activation and play a key role in regulating of apoptosis in many species(1).
Inhibitor of apoptosis proteins (IAPs):
The inhibitor of apoptosis proteins are a group of structurally and functionally similar proteins that regulate programmed cell death, cytokinesis and signal transduction(14). The IAP gene is 1.6 kb in size encoding a 31 kDa protein with a zinc finger-like motif. Many IAP family members have been identified in almost all species from viruses to mammals(15). They are characterized by the baculovirus IAP repeats (BIR) domain at the N- terminus, the name of which derives from the original discovery of these apoptosis suppressors in the genome of baculoviruses(16).
The BIR domain contains approximately 70 amino acids. Although the number of BIR domains varies among IAP members, each BIR domain is made up of cysteine and histidine residues in a well-defined pattern (CX2CX16HX6C)(15).
IAP acts as endogenous inhibitor of caspases by binding of their conserved BIR domains to the active sites of caspases in vitro and vivo. IAPs inhibit caspases by promoting the degradation of active caspases, or by sequestering the caspases away from their substrates(17).
When IAP family members are overexpressed, cancer cells no longer proceed to apoptosis and become increasingly resistant to standard chemo- and radiation therapies(18)(19). Many studies have established a circumstantial association between IAPs and cancer. Pathological overexpression of several IAP family members has been detected in several classes of human cancers(20)(21)(22).
The eight IAPs identified in humans are cIAP1, cIAP2, NAIP, Survivin, XIAP, apollon, ILP-2 and livin(23). Interestingly, many data have shown that c-IAP1, c-IAP2 and XIAP are broadly expressed in normal cells(24)(22). In normal tissues, IAPs could have some potential physiological roles, such as the regulation of the immune system(25), the response to cell damage(25), cell survival and differentiation(26). On the other hand, it has been proven in many studies that survivin, unlike other IAPs, is prominently expressed in vast majority of neoplasms but not in differentiated normal tissues(27). Survivin has been reported to be overexpressed in various cancers including breast and lung cancer, prostate, gastric, colon, bladder and esophageal carcinomas, osteosarcomas and lymphomas(28)(29). Overexpression of survivin was also found to be significantly associated with poor prognosis and decreased survivial rates in many cancers(30)(31).
Survivin:
Survivin (also Called IAP 4) is a protein with a crucial role in regulating both cell division and apoptosis. It is the smallest member of the IAP family(29). Survivin, a 16.5 kDa intracellular protein of 142 amino acid, was discovered in 1997 by Ambrosini and colleagues(32).
Structurally, survivin contains a single BIR domain. This domain is essential for its anti-apoptotic activity(33). However, instead of a ring finger domain (RING) near the C-terminus shared by others members of the IAPs, survivin contains a C-terminus alpha-helical coiled-coil (CC) domain which is thought to be important for its interaction with microtubules, hence its roles in cell cycle(34)(35)
In normal tissues, survivin shows cell -cycle dependent expression during cell division. Its expression increases in G2/M phase and decreases rapidly in G1(29). The regulation of survivn expression and function is complex and can occur at various levels, including transcriptional regulation, post-translational modification, and protein stability regulation(27). it is regulated by a number of factors such as: NF-nB(36), insulin-like growth factor I/mTOR(37), Ras oncogene family(38), E2F, Sp1, TCF, and heat shock protein (Hsp) 90(39)(40). Survivin is also regulated by p53 wild type. Additionally, post-transcriptional phosphorylation has been proven to play a regulatory role in survivin activation(41).
Biologic function of survivin
Survivin as an inhibitor of apoptosis
The mechanism by which survivin inhibits apoptosis is still controversial. Initially, survivin and other IAPs were postulated to inhibit apoptosis directly by interfering with the function of caspase-3, caspase- 7, and caspase-9(42). In support of this model, it was shown that survivin can interact with Smac/DIABLO physically, thus placing survivin in a central position in the dynamic balance of proapoptotic and antiapoptotic factors(43). However, Structural analyses of survivin indicated later that any effect on caspase should be indirect, as it lacks the amino acid sequence that is essential in other IAPs for caspase binding. Also, the survivin gene is highly conserved in a wide range of organisms, and all of its orthologues are involved in mitotic regulation but not in cytoprotection(44). Studies of cells from survivin-knockout mice have cast further doubt on the existence of a direct link between survivin and apoptosis(45).
Later experiments indicated that Survivin inhibits active caspase-9 but not active caspase-3 and caspase-7. And, survivin mediated inhibition of caspase-9 requires interaction and cooperation with other molecules such as HBXIP (hepatitis B X-interacting protein)(46) and XIAP (X-linked inhibitor of apoptosis protein) which also known as inhibitor of apoptosis protein 3 (IAP3)(47) (Fig. 3).
Survivin also provides cytoprotection to cells through the inhibition of the AIF pathway, which is known to induce caspase-independent DNA fragmentation(48).
Survivin as a promotor of mitosis
The cell- cycle dependent expression of survivin in normal tissues supports strongly its role in cell division. During mitosis, survivin acts in a narrow time window at metaphase and anaphase. It is acting as an interphase between the centromere/central spindle and the chromosomal passenger complex (CPC)(49). CPC is a hetero-tetrameric complex which localizes to different sites at different times during mitosis, and is composed of four components: Aurora-B Kinase (enzymatic component), Borealin/Dasra, Survivin and inner centromere protein (INCENP)(50)(51). CPC is essential for proper chromosome segregation and cytokinesis(52). Inactivation of mammalian survivin -or its orthologues in lower organisms – results in cytokinesis abnormalities, particularly spindle defects(53)(54) (Fig. 3)(55).
Survivin facilitating angiogenesis
In addition to it’s roles in apoptosis and mitosis, survivin promotes angiogenesis. it is strongly expressed in endothelial cells (EC) during the proliferative phase of angiogenesis(56)(57) and the antisense-mediated suppression of survivin during angiogenesis stimulates vascular regression in vitro(58). Besides, exposure of cultured vascular EC to angiogenic factors such as VEGF and bFGF result in increasing survivin expression (both mRNA and protein)(59)(60).
Survivin expression
In normal physiological conditions, survivin is usually expressed in embryonic lung and fetal organs in the developmental Stages(61). The protein is also detected in mature tissues with high proliferation potential such as thymus, placenta, CD34+ stem cells and basal colonic epithelial cells(61)(62)(63). However survivin seems to be selectively expressed in transformed cells and in most human cancers. Many studies have shown that survivin, unlike other IAPs, is prominently expressed in the vast majority of neoplasms but not in the differentiated normal tissue(27). Based on detection of protein by immunohistochemistry and mRNA by polymerase chain reaction techniques, overexpression of survivin has been reported in various human malignancies including lung cancer(64), breast cancer(65)(66); stomach(67)(68), esophagus(69), liver(70)(71), ovary cancers(72), brain(73) and hematological cancers(74).
Additionally, the immunological responses which detected against survivin supports its’ specific up-regulation in malignant cells(75)(76). Survivin protein has also been shown to induce cytotoxic T-lymphocytes (CTL) response in breast cancer, melanoma and chronic lymphatic leukemia patients(76).
Survivin expression can be deregulated in cancer by several mechanisms, including amplification of the survivin locus on chromosome 17q25 (77), demethylation of survivin exons(78), increased promoter activity(79), and increased upstream signaling in the phosphatidylinositol 3-kinase or mitogen activated protein kinase pathways(80).
Overall, increased survivin expression in several malignancies is associated with cancer survival or disease recurrence, and resistance to chemotherapy or radiotherapy. In a study of 275 patients with breast cancer demonstrated that survivin was a significant prognostic factor and predicted the outcome independent of patients’ age, tumor size and histologic grade(81). In the case of ovarian cancers, survivin expression was correlated with poor prognostic factors such as: high histologic grade, mutant p53, and poor histologic type(81)(82). Also, previous studies demonstrated that survivin was expressed in benign brain and pituitary tumors. Although survivin was also present in normal pituitary tissue, the level of the gene expression was 6-fold higher in tumors than in normal pituitary tissue(83). In a study of 222 patients who underwent radical cystectomy, survivin was expressed in 64% of bladder tumors and 94% of malignant lymph nodes, but not in normal bladder specimens and its expression correlated with disease recurrence and disease-specific mortality(84). Also, increased survivin expression has been associated with an unfavorable survival or disease recurrence in colorectal cancer(85), particularly in stage II disease in esophageal cancer(86), hepatocellular carcinoma(87), lung cancer(88), glioma(89), leukemia(90), and other cancer types. A study in oral cancer demonstrated that the extent of survivin expression was negatively correlated with the degree of differentiation(91).
Additionally, survivin overexpression may be a predictive factor to determine response to chemotherapy and radiotherapy in patients with bladder cancer(92), breast cancer(93), multiple myeloma(94), lung cancer(95) and lymphoma(96)(97). On other hand, patients with lower survivin expression were more responsive to preoperative chemotherapy with 5-flourouracil and cisplatin in esophageal cancer(98). It is also reported that patients with lower survivin expression in pretreatment biopsies were more responsive to radiotherapies in rectal cancer(99). While Overexpression of survivin was associated with resistance to a taxol-based therapy for ovarian carcinomas(100).
In addition to full-length transcript (survivin (wild type)), five splice variants, which result from splicing of survivin BIRC5 gene pre-messenger RNA (mRNA), have been described: survivin-ΔEx3, survivin-3B, survivin-2ß, survivin2α and survivin 3α with different structure and function(101)(102)(103). Previous studies showed that an imbalance in the alternative transcript ratios may affect the cell to be resistant or sensitive to apoptosis(104). This alternative splicing of Survivin has been shown to have correlation with disease activity in various patient studies. For example, studies showed that Survivin-ΔEx3 and survivin-3B were found to be highest in tumors with advanced histological grade and were associated with poor prognosis(105)(106). On other hand, the expression of survivin-2ß was significantly higher in small tumor size and was inversely associated with axillary node positive carcinomas(106).
Besides different splicing forms, immunohistochemical studies have demonstrated that survivin also localized in distinct nuclear and cytoplasmic subcellular pools. Cytosolic Survivin is believed to act as apoptotic suppressor while nuclear Survivin is postulated to regulate cell division(29). There are conflicting data of pathological significance of nuclear Survivin. Some Splicing studies showed that nuclear staining of survivin is associated with favorable prognosis(107), while others showed Its expression in the nuclei of tumor cells appears to be associated with unfavorable clinical outcomes(108)(109). Also, the cellular localization of Survivin isoforms differs. while survivin-2ß and Survivin 2a are localized in both nuclear and cytoplasmic compartments, survivin-ΔEx3 is localized in both mitochondria and nucleus(110).
Additionally, Methylation and Phosphorylation are critical requirements for survivin function. Several observations show that survivin is unmethylated in cancer but may be selectively methylate d in normal tissues with individual variations(111)(112). Methylation may play an important role in the p53 mediated suppression of survivin(113). Another critical requirement for survivin function is the phosphorylation on Thr34(114)
Treatment approaches:
Due to important role of Survivin in tumor cell division, apoptosis, chemo resistance and survival, survivin represents a unique target for biologic therapy in many human malignancies. Several novel experimental therapeutic strategies have been developed to block the expression or function of Survivin in tumour cells. These include immunotherapeutic approaches to induce immune response against Survivin, small molecule inhibitors/antagonists of survivin function, and nucleic acid based approaches which interfere with Survivin gene expression(115) such as antisense oligonucleotides (ASOs), ribozymes and small interfering RNAs (siRNAs)(116). Also, Vaccine approaches such as dendritic cell based (DC) vaccines, DNA vaccines(117), peptide vaccines for Survivin have also been evaluated in preclinical or clinical studies.
Survivin ASOs were first used against malignant melanoma cell lines. Transfection with the ASOs triggered spontaneous apoptosis linked to decreased endogenous survivin expression(118) . Treatment with LY2181308, a specific inhibitor of Survivin mRNA which has already entered the phase 1 trial(119). YM-155 is a novel small-molecule survivin suppressant which inhibits survivin mRNA transcription and protein expression in p53-deficient cancer cells in vitro(120). YM155 has also shown to be effective in vivo models of prostate, pancreatic, and lung cancer(120)(121). Ribozyme mediated approaches have also been evaluated for inhibition of Survivin expression. Down-regulation of human Survivin gene expression and increased apoptosis was achieved by using two hammerhead ribozymes (RZ-1, RZ-2) targeting human Survivin mRNA (122)