Introduction

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Extracellular cues govern the differentiation, development, proliferation, and survival of cells in multicellular organisms. Increasing evidence suggests that diffusible growth factors both direct and shape the development of organs. Limitations in growth factor availability and signaling lead to death. In fact, the availability of growth factors is thought to define the size of various tissues by dictating the delicate balance between proliferation and cell death within a particular organ (Conlon and Raff, 1999). Although normal cells require growth factor stimulation to remain viable, transformed cells often circumvent this requirement. Indeed, in many cases, tumors possess oncogenes that lead to the hyperactivation of growth and survival pathways, liberating them from the need for exogenously derived signals. Despite a wide array of distinct trophic factors and receptors that govern the survival of specific cells, many of these receptors use common intracellular signaling molecules and pathways to mediate their signals. Three pathways that have taken center stage in survival signaling are the phosphatidylinositol 3-kinase (PI3K)/Akt, the Ras/mitogen-activated protein kinase, and the Jak/signal transducers and activators of transcription (STAT) pathways. These pathways have been shown to mediate survival signals in several cell types and model organisms and in response to a diverse array of growth factors. This review will present an overview of our current understanding of programmed cell death, also referred to as apoptosis, focusing on growth factor signaling pathways in the context of cell survival and the role of these pathways in carcinogenesis. Current attempts and opportunities for therapeutic intervention are also discussed.

	Initiation and Execution of Programmed Cell Death

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The morphological features of programmed cell death were first described 30 years ago and include chromosome condensation, DNA fragmentation, and membrane blebbing. For dying cells to adopt these features, the activation of a family of cysteine proteases, termed caspases, is required. Although the activation of caspases irreversibly commits a cell to death, their activation is not required to effect cell death under many circumstances. In fact, perturbations in cell metabolism, growth factor availability, and genotoxic agents, which result in the loss of mitochondrial function, can induce cell death even in the absence of caspase activation. In the absence of mitochondrial dysfunction, however, an otherwise healthy cell may be committed to death through engagement of cell surface death receptors. Therefore, cell death can be categorized based on the method of initiation: 1) that which is initiated from cell surface death receptor engagement or 2) death arising from mitochondrial dysfunction.

Death Receptor-Induced Apoptosis. Engagement of receptors of the tumor necrosis factor receptor (TNFR) family, including CD95 (Fas/APO-1) and TNFR-1, leads to the activation of caspases to initiate death (Enari et al., 1995; Los et al., 1995). Receptor engagement then leads to the recruitment of death domain (DD) containing proteins as the Fas-associated DD, which serve to bind and activate caspase-8 that in turn ultimately initiates a cascade of caspase activation (Fig. 1). In many cell types, death receptor stimulation is sufficient to generate enough caspase activity to complete cell death. However, cells where caspase induction is not sufficient for death following CD95 stimulation require further caspase initiation generated from loss of mitochondrial function and cytochrome c release.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Cell death pathways. Apoptosis can be initiated following death receptor engagement. Alternatively, inadequate survival signaling leads to the inactivation of pro-survival Bcl-2 proteins, as Bcl-xL, and the redistribution of cytochrome c. Release of cytochrome c from the inner mitochondrial space results in caspase activation and death. See text for details. FADD, Fas-associated DD; Apaf-1, apoptotic protease-activating factor 1; PKA, cAMP-dependent protein kinase; ANT, adenine nucleotide transporter.

Cell Death following Mitochondrial Dysfunction. The critical role of the mitochondria in the regulation of apoptosis has become evident in the past several years. Cell damage that leads to perturbations in mitochondrial homeostasis and cytochrome c release from the inner mitochondrial space and into the cytosol commits a cell to death. Once in the cytoplasm, cytochrome c binds to apoptotic protease-activating factor-1, which binds and activates caspase-9 leading to initiation of the caspase cascade and cell death (Zou et al., 1997) (Fig. 1). Unlike death receptor-mediated death, most cells appear committed to dying once cytochrome c is released from the mitochondria and cannot be rescued by caspase inhibitors. The Bcl-2 family comprises proteins that are able to regulate mitochondrial homeostasis, and can be classed into two groups: those that initiate death typified by BAX and BAK, and those that function to prevent death, such as Bcl-2 and Bcl-x_L (Kelekar and Thompson, 1998). Several models have been proposed as to the biochemical function of the Bcl-2 proteins (Vander Heiden and Thompson, 1999; Desagher and Martinou, 2000). What is consistent, however, is that the expression of the pro-apoptotic Bcl-2 proteins leads to cytochrome c release and the anti-apoptotic Bcl-2 proteins serve to prevent cytochrome c redistribution.

	Growth Factors Prevent the Initiation of Apoptosis

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It is evident that growth factors prevent cell death. Inadequate growth factor signaling leads to apoptosis, and several lines of evidence suggest that the death may be attributed to loss of mitochondrial homeostasis. As detailed below, progress has been made over the past decade in identifying and elucidating growth factor signaling pathways and maintenance of viability.

PI3K/Akt Pathway. Many receptors, including those for cytokines [interleukin-3 (IL-3), IL-2], neurotrophic factors (nerve growth factor), brain-derived neurotrophic factor, and growth factors (insulin-like growth factor-1, platelet-derived growth factor) transmit survival signals through the PI3K pathway. Induction of tyrosine phosphorylation results in the activation of PI3K, which catalyzes the transfer of a phosphate group from ATP to the D3 position of phosphatidylinositol (PI), thus generating 3'-phosphatidylinositol phosphates (PIPs) (Fig. 2). PIPs have been termed lipid messengers because they serve as binding sites for proteins that possess a pleckstrin homology (PH) domain. One such protein is c-Akt (referred to through this review as Akt), also identified as protein kinase B and related to A- and C-protein kinase. Binding of the Akt PH domain to the phospholipids results in its translocation to the plasma membrane and phosphorylation at two critical residues, threonine 308 and serine 473. Phosphorylation at threonine 308 is achieved through additional kinases such as PI-dependent kinase 1, which also contains a PH domain and requires PI3K activity for membrane localization (Alessi et al., 1997). The enzyme that phosphorylates serine 473 has yet to be identified, but the Ca²⁺/CaM-dependent protein kinase kinase or the cAMP-dependent protein kinase have been suggested (Yano et al., 1998; Harada et al., 1999).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Survival signaling pathways. Three major survival pathways can be activated in response to extracellular growth factors (GF), as described in text. A schematic of downstream effectors of the Jak/STAT, PI3K, and Ras/MAPK are shown. PDK1, PI-dependent kinase 1.

Akt in Signal Transduction. The current understanding of Akt targets can be sorted into essentially two categories: proteins directly involved in signal transduction and enzymes of glucose metabolism. Among the former is BAD, a pro-apoptotic member of the Bcl-2 family. Akt phosphorylates BAD at serine 136 (S136) (del Peso et al., 1997). When phosphorylated, BAD is sequestered in the cytoplasm by 14-3-3 proteins, unable to heterodimerize with and inactivate the anti-apoptotic protein, Bcl-x_L. Overexpression of BAD alone commits a cell to death, which may be rescued by coexpression of activated Akt. However, Akt is incapable of protecting cells expressing BAD with an S136-Alanine mutation. Clearly, Akt can mediate growth factor-dependent survival by reversing the apoptotic activity of BAD. However, it is uncertain that this is the primary means of Akt protection, as Akt-dependent survival can be observed in cells that contain little to no BAD. Therefore, in these cells, Akt-mediated survival is likely to involve other mechanisms.

Akt in Metabolism Regulation. Several lines of evidence in insulin receptor signaling have emphasized the role of PI3K/Akt in cellular metabolism. Indeed, direct substrates of Akt that mediate glucose metabolism include the glucose transporter 4, phosphofructokinase 2, and glycogen synthase kinase 3 (Cross et al., 1995; Barthel et al., 1999). Interestingly, cells protected from IL-3 withdrawal-induced cell death by constitutively active Akt have high rates of glycolysis and increased mitochondrial potential while remaining viable over extended periods of growth factor deprivation. In Drosophila melanogaster, Akt null flies die during embryogenesis of systemic apoptosis. In contrast, ectopic expression of Akt during wing development results in enlarged wings composed of larger cells (Verdu et al., 1999). Recent data suggest that Akt promotes cell survival in part by maintaining elevated levels of glycolysis, thereby maintaining the cellular supply of mitochondrial substrates (Plas et al., 2001). This allows the mitochondria to use electron transport to produce the inner membrane potential that is required to maintain the integrity of the organelle in the absence of growth factor signaling. In contrast, Bcl-x_L sustains mitochondrial integrity at reduced levels of electron transport substrates. Moreover, unlike Bcl-x_L-expressing cells, the dependence on glycolysis for survival renders Akt-expressing cells susceptible to death induced from nutrient limitation. Additional studies will be required to determine whether an elevated level of metabolism or glycolysis alone is sufficient to protect cells from death in the absence of growth factors.

Ras/MAPK Pathway. Many of the same growth factors that activate the PI3K pathway can also stimulate the MAPK pathway. In addition to contributing to cell proliferation and differentiation, several studies have attributed a role in cell survival to this pathway. Upon recruitment and activation via receptor tyrosine kinases, the small guanosine triphosphatase protein Ras activates Raf, which leads to a phosphorylation signaling cascade involving the activation of the MAP kinases (Fig. 2).

Jak/STAT Pathway. The Janus family of kinases (Jaks) plays a major role in signaling from cytokine receptors to a family of STAT transcription factors (Fig. 2). The Jak/STAT pathway has been shown to transduce cytokine-mediated survival signals in several cell types. However, the mechanism is poorly understood. In some models, STAT activation leads to cell cycle arrest and cell death (Chin et al., 1996). STAT1 has been reported to trigger cell death through expression of caspase-1 (Chin et al., 1997). This apparent contradiction is probably the consequence of at least four Jak isoforms and seven STAT molecules, which may be activated by several cytokines and have distinct DNA binding properties. Knockout studies of the STAT family reveal STAT3 to have a role in cell survival (Takeda et al., 1997). STAT3 was shown to be required for growth colony stimulating factor-dependent cell survival in BaF-BO3 cell lines, and cells expressing dominant negative STAT3 were subject to apoptosis even in the presence of granulocyte factor. Downstream effectors of STAT-mediated survival are unclear, but when survival is apparent, increased Bcl-2 protein expression has been reported. Elevated Bcl-2 expression was demonstrated in cells expressing wild-type STAT3, but not dominant negative versions. However, it remains to be seen whether STAT activity is directly responsible for Bcl-2 induction.

	Pharmacological Inhibition of Survival Pathways

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Akt expression is amplified in 12% of ovarian carcinomas, 3% of breast carcinomas, and 10% of pancreatic carcinomas; PTEN mutations are found in over 80% of patients that suffer from Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome (Bellacosa et al., 1995; Cheng et al., 1996). These findings suggest the importance of the PI3K pathway in tumor progression and suppression. Activating mutations of Ras are also prevalent in 90% of pancreatic adenocarcinomas and in 50% of colon and thyroid tumors (Bos, 1989). Although the contribution of the Jak/STAT pathway in oncogenesis is still unclear, constitutive Jak activity and STAT activation is found in virally transformed cells and in leukemia (Lacronique et al., 1997). Thus, there is considerable interest in counteracting these survival pathways through therapeutic intervention, which has spawned several pharmacological agents.

Early attempts at inhibiting Ras/MAPK function focused on Ras farnesyltransferase (FTase) activity, which catalyzes the posttranslational modification of Ras, required for plasma membrane localization. Several classes of peptide and small molecule inhibitors have been discovered, some of which are highly selective for Ras FTase and have little effect on normal cells (Leonard, 1997). In fact, small molecule inhibitors are currently in clinical phase I trials (Adjei et al., 2000). The synthetic molecule PD98059 completely inhibits mitogen-activated protein kinase kinase activity in most cases and has been widely used in in vitro studies.

Although there are many pharmacological agents to study Ras/MAPK signaling, inhibition of the PI3K/Akt pathway is currently possible with two inhibitors of PI3K (Srivastava, 1998). Wortmannin is a fungal metabolite of Penicillium wortmannii that inhibits PI3K activity by irreversibly modifying K802 of the catalytic subunit. The structurally unrelated PI3K inhibitor, LY294002, is a competitive inhibitor of the ATP binding site of the catalytic subunit of PI3K. Both drugs appear to be functionally equivalent. Although successfully applied in in vitro studies, wortmannin and LY294002 have not been effectively translated to human therapy. To our knowledge, no inhibitor of the PI3K/Akt pathway has entered clinical trials.

Pharmacological activation of cell intrinsic mechanisms for death has been an attractive model for cancer therapy. Accordingly, a search for agents to induce caspases has been an objective for cancer therapeutics in recent years. Vitamin D₃ related compounds induce cell cycle arrest and apoptosis in breast cancer cells. Because Vitamin D₃ has profound effects on calcium metabolism, synthetic analogs have been sought to avoid the side effects. A phase I study with the Vitamin D₃ analog EB1089 showed reduced calcemia, and in vitro experiments demonstrate clear reduction in MCF-7 breast cancer cell line proliferation (Colston et al., 1992; Gulliford et al., 1998). Recently, EB1089 was shown to induce death by caspase-3 activation, but other mechanisms of action are possible (Mathiasen et al., 1999; Park et al., 2000). Although many extracellular stimuli induce caspase activity, such as hydrogen peroxide, radiation, and FasL, these compounds appear to induce apoptosis in both normal and transformed cells. Thus, challenges remain to specifically target caspase activation to tumors.

As mentioned, Bcl-2 family proteins have been implicated as the effectors of survival in all the major survival signaling pathways. In chemotherapy studies, Bcl-2 expression or BAX deficiency correlates with drug resistance (Minn et al., 1995). Therefore, the efficacy of any therapeutic effort to counteract hyperactivation of survival signaling needs to take the function of Bcl-2 proteins into account. In fact, Bcl-2 expression protects cells from chemotherapeutic drug-induced death (Kamesaki et al., 1993). A Bcl-2 antisense oligonucleotide approach has been investigated to reduce Bcl-2 expression and thwart its anti-apoptotic effect and has been reportedly effective in the treatment of lymphomas (Cotter et al., 1999). A phase I study using the Bcl-2 antisense molecule G3139 was shown to reduce tumor mass and circulating lymphoma cells, concomitant with a reduction in Bcl-2 expression (Webb et al., 1997; Waters et al., 2000). The preliminary evidence suggests that Bcl-2 antisense nucleotides have anti-tumor activity, with the efficacy and low toxicity similar to current chemotherapy. However, as with all antisense approaches, specific uptake and compartmentalization issues as well as questions of nonspecific nucleotide interactions will need to be addressed. In addition, further studies are required to discern the efficacy of Bcl-2 antisense therapy in nonlymphoid tissue and to sensitize cells to apoptosis in conjunction with chemotherapy.

An alternative approach to inhibiting Bcl-2 function comes from functional studies of Bcl-x_L, which suggest that the protein may promote cell survival by facilitating adenine nucleotide exchange between the mitochondria and cytoplasm. It has been proposed that Bcl-x_L may promote mitochondrial ATP/ADP exchange in the absence of growth factors by holding the voltage dependent anion channel (VDAC) in the open configuration (Vander Heiden et al., 2000). In line with this idea, an alternative method of inhibiting Bcl-2 function may be to inhibit VDAC or the associated molecule adenine nucleotide transporter.

	Concluding Remarks

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The regulation of survival has emerged as having a significant role in oncogenesis and human disease. As growth factors dictate growth, proliferation, and survival, much interest has centered on the cellular biology of growth factor signaling. Extensive use of specific inhibitors has allowed investigation of each of the pathways and their specific contribution to signaling cellular functions. Recently, however, the concept of cross-talk between the pathways has added a new dimension for study. Although there has been a greater understanding of the control of the pathways, downstream effectors of the signals remain elusive or uncertain. The PI3K pathway, for example, has several targets that may function as effectors of survival, and Akt, which is a known survival gene, has numerous direct targets that may work in preventing cell death. Furthermore, all of the pathways can influence the expression of Bcl-2 family members, which may also enhance survival. It will be necessary to learn which genes are critical in respective tissues to effectively determine which will be appropriate targets for therapeutic intervention. However, in light of recent data concerning the role of genes that prevent apoptosis in the development of malignancy, inhibition of genes involved in cancer cell survival may provide new pharmacological approaches to cancer treatment.