Clinical Cancer Research Vol. 7, 775-783, April 2001
© 2001 American Association for Cancer Research
Pharmacologic Interruption of the Mitogen-activated Extracellular-regulated Kinase/Mitogen-activated Protein Kinase Signal Transduction Pathway
Potential Role in PromotingCytotoxic Drug Action1
Paul Dent and
Steven Grant2
Departments of Medicine [S.G.], Pharmacology [S.G., P.D.], Radiation Oncology [P.D.], Physiology [P.D.], and Biochemistry [S.G.], Medical College of Virginia/Virginia Commonwealth University, Richmond, Virginia 23298
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Introduction
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Over the last 10 years, there has been a dramatic increase in interest in the role of signaling pathways in governing neoplastic cell behavior. Much of this interest has focused on members of the MAPK3
pathways, which include the ERK, the JNK, also called the SAPK, and the p38 kinase modules (1
, 2)
. Such signaling pathways regulate multiple biological activities, including cell proliferation, differentiation, cell cycle traverse, and survival, among others (3)
. Mechanistic insights into the roles of these signaling cascades in modulating neoplastic cell survival have emerged from studies investigating programmed cell death, or apoptosis. Although exceptions occur, the bulk of evidence suggests that activation of the ERK (MAPK) pathway increases, in an unknown way, the cell death threshold (4)
; conversely, activation of the JNK/SAPK and p38 kinase cascades are generally (although not universally) associated with enhanced activation of the apoptotic program (5)
. An alternative model suggests that cell-death decisions ultimately depend upon the dynamic balance between the outputs of pro- and antiapoptotic pathways. A corollary of these hypothetical models is that interruption (pharmacological or otherwise) of putatively cytoprotective signaling pathways in malignant cells could shift the balance away from survival toward cell death.
The purpose of this brief overview is not to present a exhaustive review of MEK/MAPK inhibitors; instead, it is to provide a theoretical framework for attempts to understand the possible role of such agents as modulators of cytotoxic drug action. Included is speculation concerning the downstream effectors that may be involved in this phenomenon; in addition, a summary of interactions between MEK/MAPK inhibitors and selected cytotoxic agents is presented. We refer the reader to other reviews for a broader overview of this subject (6)
.
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The "Classical" MEK/MAPK Pathway: Background
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MAPK was originally described as a Mr 42,000 insulin-stimulated protein kinase that phosphorylated the cytoskeletal protein MAP-2 (7)
. Studies by Boulton and Cobb (8)
identified an additional Mr 44,000 isoform of MAPK, which was named ERK1 (8)
. Additional studies demonstrated that the p42/p44 MAPKs regulated another protein kinase activity (i.e., p90rsk; Ref. 9
), and that they were themselves regulated by a protein kinase activity originally designated MKK (10
, 11)
.
MKK phosphorylates the MAPKs on tyrosine and threonine residues and became the first biochemically characterized dual-specificity (threonine/tyrosine) protein kinase (10
, 11)
. MKK is also often referred to as MEK. Two MKK isoforms (MKK1/2) were also found to be regulated by reversible phosphorylation, catalyzed by the proto-oncogene Raf-1 (12)
. More recently, it has been suggested that other enzymes acting at the level of MKK1/2 can phosphorylate and activate p42/44 MAPK, i.e., RIP2 (13)
, which plays a role in tumor necrosis factor-
-induced, but not epidermal growth factor-induced, MAPK activation.
Raf-1 is a member of a family of serine-threonine protein kinases consisting of Raf-1, B-Raf, and A-Raf (14)
. All "Raf" family members can phosphorylate and activate MKK1/2, although the relative ability of each member to catalyze this reaction varies (B-Raf > Raf-1 > A-Raf; Ref. 15
). Raf kinases thus act as a MAPK kinase kinase (MAPKKK). Several studies demonstrated that the NH2-terminal domain of Raf-1 could reversibly interact with the Ras proto-oncogene in the plasma membrane, and that the ability of Raf-1 to associate with Ras was dependent upon the Ras molecule residing in a GTP-bound state (16)
. Other findings demonstrated that Raf-1 activation depended upon its translocation to the plasma membrane (17)
. The regulation of Raf-1 activity is complex, with several mechanisms coordinately modulating activity when in the plasma membrane environment (18
, 19)
. Phorbol esters and the macrocyclic lactone bryostatin 1 can activate PKC, Raf-1, and the MAPK cascade in many cell types (20
, 21)
. Coincident with the demonstration of Raf-1 association with Ras, it was found that growth factors, via their plasma membrane receptors, stimulate GTP for GDP exchange in Ras using guanine nucleotide exchange factors (22)
. Thus, over an interval of 
9 years, the classical MAPK pathway has been delineated, connecting plasma membrane growth factor receptors, through guanine nucleotide exchange factors and the Ras proto-oncogene, to the Raf-1/MKK/MAPK/p90rsk module.
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MAPK versus SAPK Signaling
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The JNK/SAPK cascade was initially investigated as a pathway activated in response to cytotoxic insults. Consequently, many of the initial studies involving JNK signaling focused on the role of this pathway as either a proapoptotic or antiapoptotic effector. For example, Verheij et al. (23)
demonstrated that exposure of U937 leukemic cells to either tumor necrosis factor-, FAS ligand, ceramide, or 
-radiation activated the JNK pathway, and that this activation caused the apoptotic response to these stress signals. Multiple other studies over the past 10 years, using a large variety of cytotoxic stresses, have come to similar conclusions, i.e., that JNK activation is causally involved in proapoptotic signaling. Such conclusions are supported by evidence involving molecular inhibition of the JNK pathway at multiple levels through the use of dominant-negative mutants of MKK4/7, JNK1/2, and c-Jun (24)
. The interaction of MAPK signaling with JNK/SAPK signaling suggested a reciprocal activation/inhibition interaction in the cell death process, i.e., JNK/SAPK activation alone may not cause death, but when combined with inhibition of MAPK activity, a profound death response is elicited. Thus, the ability of MAPK signaling to modulate proliferation, differentiation, or cell death may reflect a complex summation of MAPK activity versus the activities of JNK/SAPK and p38 (23
, 24)
. It should be noted that in many of these studies, normal rather than neoplastic model systems have been used. Consequently, extrapolation of these findings to neoplastic cells exposed to cytotoxic drugs must done with caution.
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Interactions between the PKC and MEK/MAPK Pathways
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Considerable attention has been directed at the serine/threonine kinase PKC as a key modulator of the apoptotic threshold. PKC, an enzyme family consisting of at least 13 isoforms, is involved in multiple cellular functions including cell proliferation, differentiation, and stimulus-response coupling (25)
. Evidence of a role for PKC in preventing apoptosis stems from studies demonstrating that PKC activators such as phorbol myristate acetate block apoptosis in cells subjected to certain noxious stimuli (e.g., growth factor deprivation; Ref. 26
). In addition, PKC inhibitors are among the most potent initiators of apoptosis (27)
and are also capable of lowering the threshold for drug-mediated cell death (28)
. These actions stimulated interest in PKC inhibitors as possible antineoplastic agents, and some of these compounds, e.g., safingol (29)
, have entered clinical trials in humans. In this regard, the macrocyclic lactone bryostatin 1 acutely activates PKC. In contrast, after chronic exposure, bryostatin 1 down-regulates the enzyme (30)
and may mimic the actions of PKC inhibitors by potentiating drug-induced apoptosis (31)
. Multiple clinical trials involving bryostatin, either alone or in combination with established chemotherapeutic agents, have been completed or are under way (32)
. Reviews dealing with the potential role of PKC inhibitors as antineoplastic agents have appeared recently (33)
.
Although evidence that interruption of the PKC pathway promotes apoptosis in neoplastic cells is compelling, the downstream PKC targets that might be more directly responsible for this effect remain to be defined. One very plausible candidate is MAPK. As noted above, MAPK lies immediately downstream of and is activated by the dual-specificity kinases MEK1/2. MEK1/2 represent major targets of the serine/threonine kinase Raf-1. Furthermore, Raf-1 is itself activated by PKC (20)
. Consequently, inhibition of PKC (or PKC down-regulation) could ultimately reduce basal MAPK activity and/or prevent MAPK activation by diverse stimuli. Significantly, drug-induced apoptosis by agents that interrupt the PKC pathway (e.g., bryostatin 1) can be mimicked, at least in part, by pharmacological inhibitors of the MEK/MAPK cascade (e.g., PD98058; Ref. 34
). Moreover, MEK/MAPK inhibitors block the ability of PKC activators to attenuate the lethal actions of cytotoxic agents (e.g., paclitaxel; Ref. 35
). Such findings raise the possibility that agents that interrupt the MEK/MAPK pathway, such as PKC antagonists, could potentiate cytotoxic drug action.
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Pharmacological MEK/MAPK Inhibitors
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Recently, there has been a great deal of interest in using pharmacological inhibitors of MEK/MAPK to define the role of MAPK signaling in multiple cellular functions, including cell proliferation/mitogenesis, inflammatory responses, differentiation, and cell survival, among others. Much of this interest has centered on the aminoflavone MEK1/2 inhibitor, PD98059. PD98059 inhibits the activation of MEK1/2 by Raf family members at low micromolar concentrations (36)
. Other structurally unrelated compounds include U0126, also an inhibitor of the ATP binding site (37)
, and SL327, which has demonstrated MAPK inhibitory activity when given i.p. to mice (38)
. Recently, Sebolt-Leopold et al. (39)
reported that an analogue of PD98058, PD184352, inhibits MAPK activity when given p.o. in intact animals and blocks the growth of colon carcinoma cells. These and the studies cited previously raise the possibility that in addition to their intrinsic antitumor activity, MEK/MAPK inhibitors might, by shifting the balance between pro- and anti-survival signaling, enhance the lethal actions of established chemotherapeutic agents in vivo.
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Apoptosis and MEK/MAPK Signaling
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The roles of the MEK/MAPK pathway in cell survival and the regulation of apoptosis are well established (40)
. However, although our understanding of the apoptotic cascade has increased dramatically over the last several years, many aspects of this process remain enigmatic. It is currently thought that a wide variety of noxious events, including cell cycle dysregulation and cytotoxic drugs, trigger mitochondrial dysfunction (e.g., loss of the mitochondrial membrane potential; Rm) and/or release of proapoptogenic proteins (e.g., cytochrome c or apoptosis inhibitory factor) into the cytoplasm (41)
. Cytochrome c increases the activity of a multiprotein complex referred to as the apoptosome, which contains Apaf-1 (the mammalian homologue of Ced-4), dATP, and procaspase-9. This leads in turn to cleavage/activation of the effector caspase, procaspase-3 (CPP32), which is responsible for the degradation of a wide variety of critical cellular constituents, including DNA, poly(ADP-ribose) polymerase, gelsolin, actin, and pRb, among many others (42)
. Drug-induced lethality may also involve activation of the Fas/Apo-related apoptotic cascade through a pathway associated with cleavage of pro-caspase-8 (43)
. However, the ability of cytotoxic drugs to trigger this pathway appears to be highly cell type specific. Unanswered questions include how pro- and antiapoptotic family members (e.g., Bcl-2, Bcl-xL, and Bax) regulate the apoptotic process, what roles posttranslational modifications (e.g., phosphorylation) of apoptotic regulators (e.g., Bcl-2, procaspase-9) play in these events, and the in vivo functional significance of oligomerization of the apoptosome complex in cell death (44)
. Currently, the mechanism(s) by which survival (e.g., MAPK) and death (e.g., JNK/SAPK) regulate the apoptotic process remains obscure, although it is tempting to speculate that they may involve modulation of mitochondrial function, alterations in reactive oxygen metabolism, or interactions with Bcl-2 family members. Of potential relevance to this issue are recent reports that JNK may play a direct role in cytochrome c release from the mitochondria, at least in the case of certain stimuli (e.g., UV radiation; Ref. 45
), and may also be involved in Bcl-2 phosphorylation (46)
.
Although it seems plausible that signal transduction modulators act through apoptotic mechanisms, it is also recognized that apoptosis represents only one of a number of processes that can limit cancer growth, including necrosis, giant cell formation, and differentiation, among others. In addition, the extent of apoptosis, particularly at early intervals, may correlate poorly with loss of clonogenic survival (47)
. The possibility that signal transduction-active agents such as MEK/MAPK inhibitors might inhibit survival by modulating additional processes remains to be explored.
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The MEK/MAPK Module: Potential Downstream Cytoprotective Targets
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There is abundant evidence that the balance between the JNK and MAPK pathways may determine the fate of a cell after various insults (40)
. Consequently, interruption of the putatively cytoprotective MAPK module may promote cell death by shifting the balance toward stress-related events. However, it is currently unknown how MEK/MAPK signaling, or activation of downstream targets, antagonizes apoptosis. Possible distal effectors of cell survival and potential downstream targets of pharmacological MEK/MAPK inhibitors are summarized below.
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CDKIs (p21/p27).
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The CDKIs p21CIP1 and p27KIP1 inhibit the activity of cyclin/cyclin-dependent kinase complexes involved in cell cycle progression. In some cells (e.g., leukemic), such CDKIs are induced by differentiating compounds, such as phorbol esters and vitamin D3 (48
, 49)
, or in the case of p21, by DNA damage (50)
. MAPK has been implicated in the regulation of both p21CIP1 and p27KIP1 expression (51
, 52) . Furthermore, both p21CIP1 (53)
and p27KIP1 (54)
have been shown to exert antiapoptotic effects that may be distinct from their cell cycle regulatory actions. Consequently, it is possible that MEK/MAPK inhibitors promote drug-induced apoptosis by blocking basal or stimulated expression of antiapoptotic CDKIs such as p21CIP1 and p27KIP1.
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NF- B.
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The transcription factor NF-B represents a downstream target of the MEK/MAPK cascade (55)
. Although activation of NF-B has been associated with proapoptotic actions in some systems (56)
, it has also been shown to block apoptotic responses to multiple stimuli, including chemotherapeutic drugs (57)
. The antiapoptotic activity of NF-B may stem from induction of members of the inhibitors of apoptosis family (58)
.
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p90rsk; CREB.
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Recent studies indicate that cytoprotective actions downstream of Raf-1 may proceed through the Rsk family, which phosphorylates members of the CREB transcription factor family. Moreover, interruption of this pathway, e.g., by PD98059, can promote apoptosis, at least in neurogenic cells subjected to growth factor deprivation (59)
. Whether disruption of the Rsk/CREB cascade by MEK/MAPK inhibitors plays a role in cellular responses to cytotoxic drugs remains to be determined, particularly in the case of neoplastic cells.
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Bcl-2 Phosphorylation.
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Posttranslational modifications in the Bcl-2 protein, most notably phosphorylation, have been shown to play a role in regulating the response of neoplastic cells to cytotoxic drugs, particularly members of the taxane family. The bulk of evidence suggests that Bcl-2 phosphorylation contributes to taxane-mediated lethality (60)
. However, there is also evidence that in the case of certain stimuli (e.g., growth factor deprivation), Bcl-2 phosphorylation may exert an antiapoptotic effect (61)
. In this regard, MAPK has been identified recently as a Bcl-2 phosphorylating kinase that contributes to cell survival (62)
, in contrast to the effects mediated by JNK (45)
. Given the fact that there appear to be multiple phosphorylation sites within the NH2-terminal Bcl-2 phosphorylation loop, it is possible that phosphorylation of specific sites within this domain may exert disparate effects on cell survival after various noxious stimuli.
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Cell Cycle Actions.
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The effects of agents on cell cycle progression and cytotoxic drug response are closely intertwined. For example, disruption of cell cycle progression is a potent apoptotic stimulus (63)
. In addition, cycling neoplastic cells are often more sensitive to cytotoxic drugs. Consequently, signal transduction agents may influence the lethality of cytotoxic drugs on two levels, e.g., by perturbing cell cycle progression and modulating the apoptotic threshold. With respect to MEK/MAPK inhibitors, it has been shown that entry into and progression through G2-M requires an intact MAPK cascade, as does formation of the mitotic spindle (64)
. In addition, although evidence suggesting a role for MAPK in progression across the G1S boundary is less extensive (65)
, it nevertheless exists. Consequently, MEK/MAPK inhibitors may influence cytotoxic drug action through induction of cytokinetic perturbations as well as through more direct effects on apoptosis. It must be emphasized that most of the information relating G2-M progression to MAPK activation has been obtained in nontransformed cells (e.g., fibroblasts or oocytes), and the relevance of these findings to neoplastic cells remains to be definitively established.
A summary of these concepts is shown schematically in Fig. 1
, which illustrates a putatively cytoprotective signaling pathway that extends from membrane receptors and exchange factors (e.g., Grb2 and SOS) to downstream targets including MEK, MAPK, and Rsk. From a pharmacological standpoint, this pathway could be interrupted upstream at the level of Ras family members (e.g., by farnesyl or geranylgeranyl transferase inhibitors) or by PKC inhibitors (e.g., safingol) and CGP41251/PKC down-regulators (e.g., bryostatin 1). Alternatively, interruption of the pathway could take place at the level of Raf (e.g., by Raf-1 antisense oligonucleotides), or lastly, by specific MEK/MAPK inhibitors (e.g., PD98059, U0126, SL327, and PD184352). In each case, interruption of this cytoprotective pathway may lead to a lowering of the apoptotic threshold. This action could theoretically stem from interference with one or more downstream MAPK targets, including p21CIP1 or p27KIP1, CREB, or NF-B (Fig. 2)
. Alternatively, disruption of the MAPK cascade may promote apoptosis by triggering posttranslational modifications in Bcl-2, lowering levels of antiapoptotic proteins such as MCL-1 (see below), or by inducing cell cycle perturbations. These mechanisms are not mutually exclusive, and it is likely that more than one may contribute to the actions of agents that interrupt the PKC/Raf-1/MEK/MAPK cascade.
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Fig. 1. The cytoprotective MEK/MAPK pathway. Signals are transduced from the cell membrane via exchange factors that activate members of the Ras family. These, in turn, activate one or more PKC isoforms, leading to activation of Raf family members. Raf-1 phosphorylates and activates MEK1/2, resulting in phosphorylation of p42/44 MAPK and activation of multiple downstream targets, including RSK. Although exceptions have been noted, activation of this pathway is most often associated with cell proliferation and survival. Similarly, inhibitors of one or several components of this pathway block survival signals and can lead to cell death. SOS, son of sevenless; FTI, farnesyl transferase inhibitor; GGI, geranylgeranyl inhibitor; MEK1/2, MAPK kinase kinase 1/2; RSK, ribosomal S6 kinase.
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Interactions between the MEK/MAPK Cascade and Cytotoxic Drugs
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On the basis of the previous considerations, one might assume that MEK/MAPK inhibitors would uniformly enhance the lethal actions of diverse chemotherapeutic drugs. However, this is an oversimplification. Instead, the results of numerous studies suggest that the modulatory effects of MEK/MAPK inhibitors on drug action depend upon multiple factors, including drug doses, schedules, and the cell type under investigation. The following represents a brief overview of what is known about interactions between MEK inhibitors and several classes of antineoplastic agents.
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ara-C.
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ara-C is a deoxycytidine analogue that is converted to its lethal form, ara-CTP, by a series of pyrimidine salvage pathway kinases. ara-CTP inhibits DNA polymerase and is also incorporated into elongating DNA strands, leading to interference with chain initiation and elongation as well as premature chain termination (66)
. Recent studies indicate that ara-C can also act as an inhibitor of topoisomerase I (67)
. Exposure of leukemic cells to ara-C leads to generation of the lipid second messengers diacylglycerol and ceramide (68)
, the former through activation of the reverse phosphocholine transferase reaction. It is therefore not surprising that treatment of cells with ara-C can stimulate the JNK pathway (69)
as well as PKC (70)
and its downstream target, MAPK (34)
. Given the association between ceramide generation/JNK activation and cell death (23)
and the known cytoprotective effects of diacyglycerol (71)
, PKC (26)
, and MAPK (40)
, the relative outputs of these two pathways could determine the fate of a cell exposed to ara-C.
Several lines of evidence support this concept and further suggest that the activity of the MEK/MAPK pathway may be particularly critical in regulating ara-C lethality. For example, PKC inhibitors (e.g., staurosporine, H-7) lower the threshold for ara-C lethality in leukemic cell lines (28)
. This action can be mimicked by chronic (but not acute) exposure to bryostatin 1 (72)
, a potent down-regulator of PKC activity (73)
. Bryostatin 1 and the nonspecific PKC inhibitor staurosporine have also been shown to overcome, at least in part, resistance to ara-C-mediated apoptosis conferred by ectopic expression of Bcl-2 (31)
. Significantly, potentiation of ara-C-related lethality in HL-60 cells by bryostatin 1 and PKC inhibition was associated with decreased ara-C stimulation of MAPK but not of JNK activity (34)
. Moreover, the actions of bryostatin 1 were mimicked by the specific MEK/MAPK inhibitor PD98059 (34)
. Other groups have demonstrated potentiation of ara-C lethal effects by PD98059 in sympathetic neurons (74)
, whereas in one study, the cytotoxicity of low concentrations of ara-C in HL-60 cells was increased to a greater extent by the p38 MAPK inhibitor SB203580 than by PD98059 (75)
. Together, these findings raise the possibility that at least in some cell types, inhibition of a putatively cytoprotective MAPK response to ara-C exposure may lower the threshold for cell death, resulting in synergistic cytotoxicity for this drug combination.
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Taxanes.
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The taxanes, which include paclitaxel (Taxol) and taxotere, are natural products that act by inducing microtubule stabilization, disruption of the mitotic spindle apparatus, and dysregulation of mitotic events (76)
. Interference with G2-M progression caused by taxanes culminates in apoptosis (77)
. At least three separate mechanisms have been invoked to explain the lethal actions of taxanes. One theory holds that taxanes kill cells by inducing cell cycle perturbations, notably inappropriate activation of cyclin-dependent kinase 1 (p34cdc2; Ref. 78
). Alternatively, taxanes may act through a Raf-1-related mechanism to induce Bcl-2 phosphorylation, which, at least in some circumstances, promotes cell death (79)
. Finally, paclitaxel is known to trigger multiple pathways, including the JNK/SAPK and ERK/MAPK cascades (80)
. Thus, it is possible that taxane-mediated perturbations in these pathways could contribute to drug lethality.
Studies examining the impact of interruption of the MEK/MAPK cascade on paclitaxel-related cytotoxicity have yielded conflicting results, which may reflect cell-specific differences as well as schedule-dependent considerations. For example, exposure of cells to paclitaxel has been reported to result in an increase (81)
, a decrease (82)
, or no effect (83)
on MAPK activation. Consequently, effects of MEK/MAPK inhibitors on paclitaxel lethality could depend upon whether, and to what extent, MEK/MAPK activity is increased by drug exposure in a particular cell type. Furthermore, sequence-dependent considerations may also play an important role in determining the modulatory effect of MEK/MAPK inhibition on paclitaxel lethality, which may, in turn, result from cell cycle actions. As noted above, it has been shown that an intact MEK/MAPK pathway is necessary for entry into and progression through G2-M, at least in certain nontransformed cells (84)
. Consistent with this finding, Lieu et al. (85)
reported that pre- and cotreatment of human monocytic leukemic cells (U937) with PD98059 reduced paclitaxel-mediated apoptosis. In addition, simultaneous exposure of MCF-7 breast cancer cells to PD98059 opposed paclitaxel-related MAPK activation but did not modify cell survival (81)
. In marked contrast, when paclitaxel-treated U937 cells were subsequently exposed to bryostatin 1, which down-regulates PKC and its downstream target MEK/MAPK, or PD98059, which directly inhibits MEK/MAPK, paclitaxel-related apoptosis was enhanced (86)
. Consistent with the latter results, PD98059 attenuated the ability of phorbol myristate acetate to block paclitaxel lethality in HL-60 cells (35)
. Moreover, it has been shown very recently that the MEK inhibitor U0126 enhanced paclitaxel-mediated apoptosis in breast, ovarian, and lung cancer cells (87)
, a finding compatible with the results of Wang et al. (86)
. One plausible explanation for these disparate findings is that pretreatment of cells with MEK/MAPK inhibitors may oppose entry of cells into the paclitaxel-sensitive G2-M phase of the cell cycle, whereas subsequent exposure of paclitaxel-treated cells to MEK/MAPK inhibitors might prevent escape from G2-M, leading to an enhanced apoptotic response. An alternative possibility is that the impact of MEK/MAP kinase inhibitors on paclitaxel-associated lethality may depend upon the net effect on induction of the CDKI p21CIP1, which can oppose taxane-related apoptosis (88)
. For example, in cells in which paclitaxel fails to stimulate MAPK-associated p21CIP1 induction, lethality may prove to be MAPK independent (89)
. It remains to be determined whether these sequence-dependent interactions can be extended to include solid tumor cells, as well as other MEK/MAP kinase inhibitors.
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Cisplatin.
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Recently, there has been considerable interest in defining the functional role of MAPK1/2 in cisplatin-mediated cytotoxicity. The existing evidence that exists suggests that it plays a cytoprotective role. For example, Persons et al. (90)
reported that exposure of two ovarian carcinoma cell lines (e.g., SK-OV-3 and UCI 101) to cisplatin resulted in MAPK activation, whereas transplatin, which was not cytotoxic to these cells, did not. Moreover, treatment of cells with PD98059 effectively blocked cisplatin-mediated MAPK activation and increased cisplatin-related lethality. In A2780 ovarian carcinoma cells, potentiation of cisplatin lethality by PD98059 was linked to interference with p53 phosphorylation (91)
. Thus, the putative cytoprotective effects of MAPK activation accompanying cisplatin exposure is similar in many respects to that observed in the case of ara-C (see above). In accord with these findings, Hayakawa et al. (92)
reported that cisplatin differentially activated ERK and JNK in the ovarian carcinoma cell lines A2780 and Caov-3, and that PD98059 promoted cisplatin-related lethality in both cell types. Together, these observations suggest that induction of DNA damage by cisplatin triggers MAPK activation and antagonism of cell death. They also indicate that pharmacological interruption of this pathway can lower the threshold for cisplatin-mediated lethality. Interruption of the JNK pathway has also been shown to promote cisplatin-related toxicity, possibly by interfering with DNA repair events (93)
.
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Topoisomerase II Inhibitors.
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A systematic evaluation of the effects of MEK/MAPK inhibition on the response of tumor cells to topoisomerase II inhibitors such as doxorubicin in neoplastic cells has not as yet been performed. Zhu et al. (94)
reported that doxorubicin-mediated free radical production elicited an ERK1/2 response in normal cardiac myocytes, and that interruption of this pathway by PD98059 enhanced doxorubicin-related apoptosis. It remains to be determined whether pharmacological MEK/MAPK inhibitors would have a similar (or greater) effect in neoplastic cells, and if so, whether a net increase in the therapeutic index of doxorubicin might ensue. In this regard, treatment of KB-3 carcinoma cells with doxorubicin failed to induce ERK activation (95)
, although effects of agents such as PD98059 on doxorubicin-related toxicity have not been fully characterized in this system.
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Topoisomerase I Inhibitors.
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Little information is available concerning the relationship between MEK/MAPK activation and the apoptotic response of neoplastic cells to topoisomerase I inhibitors. However, Hetman et al. (96)
reported that the protective effects of brain-derived neurotropic factor against the toxicity of the topoisomerase I inhibitor, camptothecin, in normal cortical neurons was mediated through the ERK pathway. They found that in these cells, coadministration of PD98059 increased camptothecin-related lethality. As in the case of topoisomerase II inhibitors, the net effect of MEK/MAPK inhibition on the therapeutic index of agents such as camptothecin remains to be determined.
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Vinblastine.
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Recent studies demonstrated that microtubule-active agents such as vinblastine induce ERK activation (95)
, and that ERK activation is critical to up-regulation of the antiapoptotic protein MCL-1, at least in ML-1 human leukemia cells (97)
. Furthermore, MEK/MAPK inhibitors are capable of blocking the increase in MCL-1 expression, leading to a lowering of the apoptotic threshold. Thus, in addition to perturbing Bcl-2 phosphorylation (62)
, interruption of the MEK/MAPK pathway may also modify the expression of other apoptotic regulatory proteins in tumor cells and thereby influence drug sensitivity.
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Summary and Future Directions
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The MEK/MAPK pathway represents an attractive target for therapeutic intervention. The development of pharmacological inhibitors of this pathway, particularly those exhibiting in vivo activity, remains an area of intense interest. On the basis of preclinical evidence, it seems reasonable to propose that MEK/MAPK inhibitors can sometimes mimic the actions of PKC inhibitors in lowering the threshold for cytotoxic drug-mediated cell death. Although the notion that PKC inhibitors act primarily by interfering with MEK/MAPK is an oversimplification, it seems likely that the PKC/Raf-1/MEK/MAPK cascade plays an important cytoprotective role in at least some tumor cell types, and that interruption of this pathway could increase the cytotoxic activity of a number of clinically effective agents. The ability of MEK/MAPK inhibitors to promote drug-induced lethality appears to be cell type specific, as well as dose and schedule dependent. Optimization of the strategy of combining MEK/MAPK inhibitors with established chemotherapeutic drugs will require resolving a number of questions. These include: (a) identifying specific downstream MEK/MAPK targets responsible for cytoprotective actions; (b) defining the relationship between the chemopotentiating actions of MEK/MAPK inhibitors and their effects on cell-cycle progression; (c) understanding the mechanism(s) underlying sequence-dependent interactions between MEK/MAPK inhibitors and cytotoxic drugs; and (d) determining whether synergistic in vitro interactions between MEK/MAP kinase inhibitors and chemotherapeutic drugs occur in animals. The latter studies should be facilitated by the development of newer MEK/MAPK inhibitors with enhanced in vivo activity. Currently, studies examining interactions between MEK/MAPK inhibitors have been restricted to a relatively limited number of established chemotherapeutic agents. Hopefully, expansion of such efforts to include a wider range of clinically relevant agents will help answer these questions and lead to the development of a new chemotherapeutic strategy in which cytotoxic drugs are combined with novel signal-transduction inhibitors.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants CA 63753, CA 83705, CA 72955, and DK 52825 from the NIH and Grant BC98-0148 from the Department of Defense. 
2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Medical College of Virginia/Virginia Commonwealth University, MCV Station Box 230, Richmond, VA 23298. Phone: (804)828-5211; Fax: (804)828-8079; E-mail: stgrant{at}hsc.vcu.edu
3 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular receptor kinase (extracellular signal-regulated kinase); JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; MEK, mitogen-activated/extracellular-regulated kinase; MKK, MAPK kinase (MEK); PKC, protein kinase C; CDKI, cyclin-dependent kinase inhibitor; NF-B, nuclear factor-B; CREB, cAMP-responsive element binding protein; ara-C, 1-ß-D-arabinofuranosylcytosine.
Received 9/ 8/00;
revised 12/12/00;
accepted 1/ 6/01.
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Introduction
The "Classical" MEK/MAPK...
m). Interference with one or more of these events by MEK/MAPK inhibitors may, depending upon the cell context, lower the threshold for certain forms of drug-induced lethality.