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Address for correspondence: Kristina Viktorsson, Unit of Medical Radiation Biology, Department of Oncology-Pathology, Cancer Centrum Karolinska R8:00, Karolinska Institutet, SE-171 76 Stockholm, Sweden
Tumor cells may respond to chemo- or radiotherapy by activation of several cellular signaling cascades that influence cell survival and cell death, including activation of cell cycle arrest, senescence or triggering of several cell death types (i.e., mitotic catastrophe, necrosis, or apoptosis).
However, tumor cells derived from solid tumors are often refractory to therapy or develop resistance during the treatment course. This is illustrated by non-small cell lung cancer (NSCLC), which shows a high degree of intrinsic resistance, and by small cell lung cancer (SCLC), which often develops resistance to treatment during the course of disease.
However, it is also likely that other cell death modes (e.g., necrosis, autophagy, and mitotic catastrophe) and premature senescence are of equal importance for efficient tumor cell death in response to chemo- and radiotherapy.
In this article, we give a brief overview of the main apoptotic signaling pathways and their deregulation in lung cancer (LC), and we provide some examples of apoptosis-based therapies.
Apoptosis is distinguished by some morphological characteristics (i.e., plasma membrane blebbing, cell shrinkage, condensation/fragmentation of the chromatin, and disintegration of the cell into apoptotic bodies). All these characteristics are effects of selective proteolysis of proteins involved in cell signaling, DNA repair, or structural maintenance of DNA integrity, carried out by caspases, a group of cystein-aspartate enzymes.
Caspases are classified as initiator caspases (caspase-2, -8, -9, and -10 within human cells), which, upon activation, cleave and activate the second group, the effector caspases (mainly caspase-3, -6, and -7 within human cells), then perform selective proteolysis.
Caspases are activated either by death receptor (DR) activation (extrinsic) or via mitochondrial release of apoptogenic proteins (e.g., cytochrome c, smac/DIABLO, and HtrA2/Omi) (intrinsic) (Figure 1). The signals propagated by the intrinsic pathway may also be generated in cell nuclei or lysosomes or within the endoplasmatic reticulum (Fig. 1).
FIGURE 1Apoptotic signaling pathways. Caspases are activated by extrinsic (death receptor-mediated) or intrinsic pathway (mitochondria-mediated) signaling. Death ligands bind to their receptors (Fas-L and Fas-R) and via death domain (DD) adaptor proteins (FADD) are bound. Via a death effector domain (DED), the adaptor protein recruits pro-caspase-8, which gets activated. Caspase-8 thereafter directly activates pro-caspase-3, which cleaves structural proteins and inhibitor of caspase-activated DNase (ICAD), resulting in free caspase-activated DNase (CAD), which causes fragmentation of nuclear DNA. To amplify the signal, caspase-8 may also cleave Bid into t-Bid, which can initiate mitochondria-mediated signaling. The mitochondria-mediated pathway results in increased mitochondrial outer membrane permeability (MOMP) and release of apoptogenic proteins to cytosol (cytochrome c and Smac/DIABLO). The Bcl-2 family proteins (Bcl-2, Bcl-XL, Bak, Bax, Bad, and Bid) in part control MOMP. Within cytosol, cytochrome c forms a complex together with Apaf-1 the apoptosome, in which pro-caspase-9 is activated. Activated caspase-9 then triggers pro-caspase-3 activation. At several levels, caspase processing and/or activity can be inhibited by inhibitor of apoptosis proteins (IAPs) or by heat shock proteins (HSPs). IAPs are antagonized by Smac/DIABLO. Apoptotic signaling is also influenced by growth factor receptor signaling (exemplified by the Akt-pathway), which blocks Bad function by inducing binding to 14-3-3 proteins. Apoptotic signals can also be initiated at other places within the cell, (exemplified by the cell nuclei), in which p53, PUMA, NOXA, and caspase-2 gets activated on DNA damage and transmit pro-apoptotic signals to mitochondria.
In the extrinsic caspase activation pathway, TNF superfamily ligands bind to DRs, causing oligomerization of DRs and recruitment of adaptor proteins via a death domain. In turn, adaptor proteins bind pro-caspase-8 via a death effector domain (DED) allowing pro-caspase-8 to be activated, an event that is critically dependent on the adaptor recruitment domain in the pro-caspase-8.
In the intrinsic pathway, apoptotic signals trigger increased mitochondrial outer membrane permeability (MOMP), followed by selective release of apoptogenic proteins from the mitochondrial inter membrane space to the cytosol (e.g., cytochrome c, Smac/DIABLO, and HtrA2/omi), all which promote caspase activation (Fig. 1).
Cytosolic cytochrome c forms a complex with apoptosis protease-activating factor 1 (Apaf-1) and dATP (i.e., the apoptosome), in which the dimerization of pro-caspase-9 occurs, allowing its activation into caspase-9. This is followed by pro-caspase-3 activation.
For these to efficiently result in apoptotic propagation, the concomitant alleviation of the caspase-blocking effect of inhibitor of apoptosis proteins (IAPs) is required. Hence, the release of Smac/DIABLO and HtrA2/omi, both which block IAPs and both which are released as a consequence of increased MOMP, leads to increased caspase-3 activity.
In part, MOMP is controlled by Bcl-2 family proteins, and the anti-apoptotic members Bcl-xL and Bcl-2 both inhibit MOMP. Accordingly, pro-apoptotic members such as Bak or Bax, both which are activated by some BH3-only proteins (Bid, Bim, Bad, PUMA, and NOXA), can promote MOMP.
An important regulator of mitochondria-mediated signaling is the tumor suppressor p53. Thus, p53 may induce expression of pro-apoptotic proteins (e.g., Bax, PUMA, Apaf-1) and/or repress anti-apoptotic proteins, including Bcl-2, in response to DNA damage.
Caspase activity can be restrained by inhibitor of apoptosis proteins (IAPs), by heat shock proteins (HSPs), or by changes in protein kinase signaling. Briefly, IAPs (cIAP-1,-2, XIAP, and survivin) cause a structural block within the substrate-binding pocket of caspases, which impede substrate binding and target the bound caspases for proteosomal degradation.
HSPs (HSP90, HSP70, HSP60, and HSP27) can block caspase activity through sequestration of cytochrome c, inhibition of Bid redistribution, or Akt dephosphorylation or by blocking Apaf-1–mediated pro-caspase-9 or -3 activation.
The phosphatidylinositol 3-kinase (PI3-K)/Akt-dependent pathway and the Ras-activated mitogen-activated protein kinase (MAPK) pathway both influence apoptotic propensity.
Although Akt and MAPK/ERK mainly are activated by growth factors and inhibit pro-apoptotic signaling, the MAPKs p38 and JNK can also be activated in response to cellular stress (e.g., DNA damaging treatments) and be either pro- or anti-apoptotic depending on stimuli, duration, and cell type.
Activation of Bak and Bax through c-abl-protein kinase Cdelta-p38 MAPK signaling in response to ionizing radiation in human non-small cell lung cancer cells.
In a patient with NSCLC, material consisting of approximately 100 specimens, somatic mutations of TRAIL receptor 2 were found in approximately 10% of the patients.
However, if the mutations in TRAIL receptor 2 influenced the patient response to chemo- or radiotherapy or overall survival remains to be examined. Decreased expression of Apaf-1 was reported in NSCLC tumors compared with normal lung, whereas pro-caspase-9 and -3 were up-regulated.
With respect to Bcl-2 family proteins, we reported that radioresistant NSCLC cells display no or little Bak or Bax activation compared with radiosensitive NSCLCs or SCLCs.
Briefly, c-IAP-1, XIAP, and survivin were reported to be differentially expressed in a panel of SCLC and NSCLC cell lines in a non-tumor type-dependent manner.
However, in a clinical LC material, c-IAP-1, -2, and XIAP were reported not to correlate to clinically related prognostic factors (e.g., tumor size, stage, histology, and grade) or to tumor chemotherapeutic response.
When examining HSP27 and HSP70 in NSCLC clinical specimens, expression was found in 60% of the cases, and HSP70 expression was correlated to histopathological differentiation, clinical stages, smoking history, or lymph node metastasis.
It is well established that lung cancer, especially NSCLC, is driven by increased growth factor signaling. Thus IGF-1R, EGF-R (erbB1), or K-Ras are all often over-expressed or constitutively active in NSCLC and/or SCLC and may cause increased anti-apoptotic signaling.
Moreover, we have also shown that deficiency in activation of MAPKs such as JNK and/or p38 may also contribute to impaired radiation-induced apoptotic responses.
Several concepts of increasing apoptotic signaling as a way to improve chemo- and radiotherapy responses have been introduced and are in preclinical development to allow clinical use or have entered into clinical trials.
Instead, the TNFα-related apoptosis, inducing ligand TRAIL, which bind to TRAIL-R1 (DR4) and TRAIL-R2 (DR5/Killer), has shown promising results in the recombinant form alone or together with chemo- or radiotherapy.
recently showed a radiosensitizing effect involving induction of apoptotic signaling, inhibition of tumor growth, and prolonged survival of the tumor-bearing mice.
In preclinical lung cancer models, alleviation of IAP function using either antisense or siRNA or peptides mimicking the endogenous IAP inhibitor Smac has been tested either alone or in combination with chemo- or radiotherapy and have, to some extent, been promising.
Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide.
Strategies in which Bcl-2/Bcl-xL expression is inhibited or BH3-mimetics applied have also been introduced. Thus, antisense against Bcl-2 (Oblimersen) has been tested in NSCLC and other tumor types and has reached phase III trials.
Mutations in the p53 gene, which impede its function as a transcriptional regulator of apoptosis, are common in both NSCLC and SCLC (50% and 70%, respectively).
Hence, one alternative to revert chemo- or radiotherapy resistance in lung cancer may therefore be to restore p53 function. This has been clinically tested in NSCLC by using wildtype-p53 gene transfer either alone or in combination with chemotherapy.
However, the clinical usefulness of such an approach awaits further studies.
ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Cancer Society (to RL), the Stockholm Cancer Society (to RL and KV), and the Funds of the Karolinska Institutet.
REFERENCES
Johnstone RW
Ruefli AA
Lowe SW
Apoptosis: a link between cancer genetics and chemotherapy.
Activation of Bak and Bax through c-abl-protein kinase Cdelta-p38 MAPK signaling in response to ionizing radiation in human non-small cell lung cancer cells.
Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide.
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