Proapoptotic BH3-Only BCL-2 Family Protein BIM Connects
Death Signaling from Epidermal Growth Factor Receptor
Inhibition to the Mitochondrion
Jing Deng, Takeshi Shimamura, Samanthi Perera, Nicole E. Carlson, Dongpo Cai,Geoffrey I. Shapiro, Kwok-Kin Wong, and Anthony Letai
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
Abstract
A subset of lung cancers expresses mutant forms of epidermalgrowth factor receptor (EGFR) that are constitutively activated.Cancers bearing activated EGFR can be effectively targetedwith EGFR inhibitors such as erlotinib. However, the death-signaling pathways engaged after EGFR inhibition are poorlyunderstood. Here, we show that death after inhibition of EGFRuses the mitochondrial, or intrinsic, pathway of cell deathcontrolled by the BCL-2 family of proteins. BCL-2 inhibitscell death induced by erlotinib, but BCL-2–protected cells arethus rendered BCL-2–dependent and sensitive to the BCL-2antagonist ABT-737. BH3 profiling reveals that mitochondrialBCL-2 is primed by death signals after EGFR inhibition inthese cells. As this result implies, key death-signaling proteinsof the BCL-2 family, including BIM, were found to be up-regulated after erlotinib treatment and intercepted by over-expressed BCL-2. BIM is induced by lung cancer cell lines thatare sensitive to erlotinib but not by those resistant. Reductionof BIM by siRNA induces resistance to erlotinib. We show thatEGFR activity is inhibited by erlotinib in H1650, a lung cancercell line that bears a sensitizing EGFR mutation, but thatH1650 is not killed. We identify the block in apoptosis in thiscell line, and show that a novel form of erlotinib resistance ispresent, a block in BIM up-regulation downstream of EGFRinhibition. This finding has clear implications for overcomingresistance to erlotinib. Resistance to EGFR inhibition can bemodulated by alterations in the intrinsic apoptotic pathwaycontrolled by the BCL-2 family of proteins. [Cancer Res2007;67(24):11867–75]
Introduction
Lung cancer is the leading cancer-related cause of death in theUnited States. Reversible anilinoquinazoline tyrosine kinaseinhibitors (TKI) targeting the epidermal growth factor receptor(EGFR), such as erlotinib and gefitinib, have shown clinical activityin patients with non–small cell lung cancer (NSCLC; refs. 1, 2).Activating point mutations and deletion mutations in the kinasedomain of EGFR have been found to correlate with response tothese TKIs (1–3). Mutations fall into four major classes: single-basesubstitutions in exon 18; deletions in exon 19; insertion/
duplications in exon 20; and a single-base substitution, L858R,in exon 21 (4–6). Although EGFR inhibitors produce dramaticresponses in this population, acquired resistance to TKIs invariablyemerges over time, in part mediated by a substitution mutationin exon 20 (T790M), known to confer resistance to gefitinib orerlotinib (7, 8). Among other mechanisms, this mutation caninduce steric hindrance that limits access of drug to the ATP-binding pocket in the EGFR kinase domain. Recently, theemergence of c-Met amplification during the course of EGFR-target therapy has been identified as another novel resistancemechanism to reduce the efficacy of gefitinib (9). To overcome theresistance of cells expressing EGFRs harboring kinase domainmutations with the secondary T790M mutation, irreversible TKIs,such as CL-387,785 and HKI-272 have been used (10, 11), withvariable potency in cell culture systems.
Most importantly, not all NSCLC patients with EGFR mutationsinitially respond to EGFR-targeting therapy, suggesting there aresubsets of NSCLC cases where unknown underlying mechanismsare responsible for resistance under the complete inhibition ofEGFR-signaling axis by kinase inhibitors. Furthermore, in the caseof acquired resistance, after accounting for c-MET amplificationand T790M EGFR mutations, there remains f30% of resistantcases in which the molecular basis for resistance remains to beelucidated. It seems likely that many of these are resistant due toalterations in signaling pathways downstream of inhibition ofEGFR activity, including whatever death-signaling and executingpathways are used. An important signaling pathway whose role ispoorly understood in EGFR targeting is the programmed cell death(PCD) pathway. Therefore, we turned our attention to determininghow EGFR inhibition was signaled to and executed by PCDpathways.
Apoptosis is the most thoroughly studied form of PCD; mostchemotherapeutic agents can kill via apoptotic pathways. Apop-tosis can progress along two distinct pathways, the extrinsic andthe intrinsic (12). The extrinsic pathway is activated after ligationof any of several of the cell surface receptors in the TNFR family.After ligation, a death complex forms that results in the activationof the initiator caspase 8, which in turn can activate effectorcaspases, such as caspase 2 and caspase 7, resulting in widespreadproteolysis, cellular dysfunction, and commitment to PCD.
The intrinsic pathway is also known as the mitochondrialapoptotic pathway because commitment to death via this pathwayhinges on the control of permeabilization of the mitochondrialouter membrane. This pathway is controlled by interactions amongthe proapototic and antiapoptotic members of the BCL-2 familyof proteins. In response to a wide range of signals of damage ordysfunction, including DNA damage, microtubule disruption, orgrowth factor withdrawal, certain of the proapoptotic BH3-onlyclass of BCL-2 proteins are up-regulated. Up-regulation can occur
Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).
K. Wong and A. Letai contributed equally to the work.Requests for reprints: Anthony Letai, Dana 530B, Dana-Farber Cancer Institute,
via an increase in protein levels, a change in subcellularlocalization, or acquisition of a posttranslational modification.These changes, in turn, can be caused by mechanisms includingtranscriptional means, phosphorylation, protease cleavage, or byprotein stabilization (13, 14). The activator subtype of BH3-onlyproteins, which includes at least BID and BIM, can then proceedto activate proapoptotic BAX or BAK (15–18). Activated BAX andBAK undergo an allosteric change and oligomerize, and subse-quently participate in the formation of a pore that permeabilizesthe mitochondrial outer membrane (19–22). Proapoptotic con-tents, including cytochrome c , apoptosis-inducing factor, andSMAC/Diablo, are exposed to the cytoplasm where they engagevarious aspects of the downstream apoptotic machinery. Forinstance, cytochrome c forms a complex with caspase 9, apoptosisprotease-activating factor 1, and ATP to form the apoptosome,which then activates effector caspase 3 (23). Permeabilization ofthe mitochondrial outer membrane may be considered the point ofcommitment to PCD, the point of no return. BCL-2, along withrelated cellular antiapoptotic proteins BCL-XL, BCL-w, BFL-1, andMCL-1 interfere with the progression of death signals by bindingand sequestering activator BH3-only proteins, preventing theiractivation of BAX and BAK (15, 24–26). These antiapoptoticproteins may also prevent death by binding BAX and BAK,particularly their activated forms (27). This system is furthermodulated by the sensitizer BH3-only proteins that lack the abilityto activate BAX or BAK but nonetheless exert their prodeathfunction by competing for the BH3 domain–binding site inantiapoptotic proteins such as BCL-2 (15, 17). In doing so, theycan displace activator proteins, facilitating the death cascade.Examples of sensitizer BH3-only proteins include BAD andNOXA. Notably, the sensitizer BH3-only proteins exhibit a selectivepattern of interaction with antiapoptotic proteins, so that eachantiapoptotic protein may be distinguished functionally based onits pattern of interaction with the BH3 domains of sensitizer BH3-only proteins (15, 16, 28, 29).
The intrinsic, or mitochondrial, pathway of PCD plays animportant role in killing cancer cells in response to many types oftherapies. Yet it is generally poorly understood how the initialevent, drug reaching target, is connected molecularly to theproteins that control commitment to mitochondrial cell death, theBCL-2 family of proteins. We investigated whether the intrinsicpathway of PCD is essential for death after inhibition of mutatedEGFR. We found that the intrinsic pathway was indeed essentialand, furthermore, show that BIM is a key mediator of the deathsignal. This knowledge allows us to identify a novel mechanism forerlotinib resistance downstream of EGFR inhibition but upstreamof BIM up-regulation. Such placement is the first step in investi-gation of strategies to overcome resistance to erlotinib.
Materials and Methods
Cell lines. Cell lines described in this paper (unless specified otherwise)
were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-
(IL-3)–dependent murine pro-B Ba/F3 cells were transduced withretrovirus containing the EGFR L858R mutant as describedpreviously (30). This prior study had shown that Ba/F3 cellsexpressing the L858R mutant were able to grow independently ofIL-3, but that treatment of such cells with an inhibitor of EGFRinduced cell cycle arrest and apoptosis (30, 31). Other studiesshowed that in these Ba/F3 cells, erlotinib induced dephosphor-ylation of EGFR and of downstream targets of EGFR (32). Toexamine the molecular mechanisms involved in the apoptosisinduced by this targeted therapy, we examined the effects of BCL-2expression, as BCL-2 inhibits initiation of the mitochondrial, orintrinsic, pathway of apoptosis. We transfected Ba/F3 cellsexpressing L858R with BCL-2 or empty vector control (Fig. 1A).
Empty vector-transfected cells treated with erlotinib were killedas measured by Annexin V staining. Annexin V stains cells in whichthe phosphatidyl serine on the inner leaflet of the plasmamembrane is externalized, a finding consistent with apoptoticdeath. BCL-2 inhibited cell death initiated by erlotinib, demon-strating that the mitochondrial apoptotic pathway is required forerlotinib-induced death (Fig. 1B, top).
An important mechanism by which BCL-2 inhibits cell death isby sequestering prodeath molecules of the BCL-2 family. Thissequestration relies on binding of the BH3 domain of the prodeathproteins. ABT-737 is a small molecule mimetic of the proapoptoticBH3 domain that can be used as a competitive inhibitor ofantiapoptotic BCL-2 function. Treatment with ABT-737 can
displace prodeath BH3-containing molecules from BCL-2, allowingfor the progression of death signaling. We hypothesized that ifBCL-2 is protecting from cell death by binding proapoptotic BH3-containing proteins generated by EGFR inhibition, then ABT-737should reverse the protection afforded by BCL-2. Vector or BCL-2–transfected cells were resistant to ABT-737 treatment in theabsence of erlotinib (Fig. 1B, middle). However, ABT-737 treatmentreversed the protection from erlotinib afforded by BCL-2 (Fig. 1B,bottom). Comparison with Fig. 1B (top) reveals that treatment with100 nmol/L ABT-737 restored sensitivity to erlotinib in BCL-2–expressing cells to that of vector-transfected cells. Hence, we hadfound that treating a cell expressing an activating mutant of EGFRwith erlotinib induced death via the mitochondrial apoptoticpathway using BH3 domain-containing proapoptotic proteins.BCL-2 could prevent this death, at the expense of rendering thecell newly dependent on BCL-2 function. Protection by BCL-2 couldthen be abrogated and dependence exploited by treatment with theBCL-2 antagonist ABT-737.
EGFR can be activated by several different mutations. To testwhether this pathway of death signaling was specific to the L858Rmutant, or rather more generalizable to EGFR activation in general,we turned to a functionally distinct activating mutant of EGFRcontaining a deletion. L747-P753del7insS (del4) is one of the mostprevalent deletion mutations found in NSCLC patients. Wetransfected Ba/F3 cells previously transduced with the deletionwith Flag-BCL-2 or empty vector control (Fig. 1C). Similar to cells
Figure 1. BCL-2 blocks erlotinib killing viamitochondrial apoptotic pathway; ABT-737restores sensitivity to erlotinib. A, Westernblot analysis of FlagBCL-2 and EGFRexpression levels in L858R BA/F3 mutantclones transfected with control vector (vec ) orpCI-Neo.FlagBCl-2 (bcl-2 ). Endogenousmouse BCL-2 was also shown. B, cellviability of L858R BA/F3 vec or bcl-2 cellstreated with increasing doses in erlotinib(top ), ABT-737 (middle ), or the combinationof erlotinib and ABT-737 (100 nmol/L;bottom ) after 48-h incubation withcompound(s). Cells were stained withfluorescent conjugates of Annexin V for flowcytometry analysis. Viability was shown as apercentage of control (DMSO treated) cells;error bars, SD of experiments done intriplicate. C, Western blot analysis ofFlagBCL-2 and EGFR expression levels inDel4 BA/F3 mutant clones transfected withthe following control vectors: vec (3), vec (4)or pCI-Neo.FlagBCl-2 [bcl-2 (9) ], and bcl-2(13) . Endogenous mouse BCL-2 in thosecells was also shown. D, cell viability assaysperformed in Del 4 BA/F3 cells (clone typesas indicated) as in B with increased doses inerlotinib (top ), ABT737 (middle ), or thecombination of erlotinib (increased doses)and ABT-737 (100 nmol/L; bottom ).
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transduced with L858R, cells transduced with the del4 mutant werekilled by erlotinib (Fig. 1D, top), a death that was in turn inhibitedby BCL-2. Protection by BCL-2 was again abrogated by treatmentwith the BH3-mimetic ABT-737 (Fig. 1D, middle and bottom). Thus,we concluded that death signaling via the intrinsic apoptoticpathway is likely a general property of erlotinib inhibition ofactivated EGFR, not restricted to a specific activating mutation.
We hypothesized that BCL-2 was preventing death by seques-tering prodeath signals at the mitochondrion. To test thishypothesis, we used a strategy called ‘‘BH3 profiling’’ that detectsthe priming of BCL-2 with death signals at the mitochondrion (15).We have previously shown that cells in which BCL-2 is activelyintercepting death signaling by binding and sequestering prodeathBH3-only proteins may be considered ‘‘primed for death’’ (15, 33).Such cells are dependent on BCL-2 function for survival. Thisdependence can be detected using BH3 profiling.
BH3 profiling tests mitochondria from the cell of interest byexposing them to peptides derived from the BH3 domains ofprodeath BH3-only molecules. Only mitochondria bearing anti-apoptotic proteins ‘‘primed’’ with activator BH3 death signalsrespond to sensitizer BH3 peptides, as detected by cytochrome crelease. Moreover, the pattern of response to sensitizer peptidesmay be compared with the binding pattern of the individualantiapoptotic proteins to determine exactly which antiapoptoticproteins are bearing the activator BH3-only proteins. Thistechnique has been described in more detail elsewhere (15, 33).If our model is correct, then we would expect mitochondria toshow limited dependence on BCL-2 when the cell was expressingactivated forms of EGFR in the absence of erlotinib, with orwithout BCL-2. However, we would expect that cells expressingactivated EGFR and BCL-2 would show increased dependence onBCL-2 after erlotinib treatment, as the mitochondrial BCL-2 would
Figure 2. Cellular BCL-2 dependenceafter EGFR inhibition results from‘‘priming’’ of BCL-2 at the mitochondrion.Mitochondria were isolated from differentEGFR mutants cells and incubated with apanel of BH3 peptides (100 Amol/L),ABT-737, or enantiomer (concentrations asindicated). Release of cytochrome c wasdetermined by a comparison of cytochromec in the pellet and supernatant quantitatedby ELISA. Values shown reflect subtractionof background determined fromDMSO-treated samples and normalized torelease by BIM; error bars, SD of duplicateexperiments. A, B,and C, BH3 profiling ofL858R BA/F3 cells (left) bearing withvector-only (A, left), bcl-2 (untreated;B, left ), or bcl-2 treated with 0.2 Amol/Lerlotinib for 48 h (C, left ), respectively.A, B,and C, BH3 profiling of Del 4 BA/F3cells (right ) bearing with vector-only(A, right ), bcl-2 (untreated; B, right ), orbcl-2 treated with 0.1 Amol/L erlotinib for48 h (C, right ), respectively. Note thaterlotinib treatment ‘‘primes’’ BCL-2expressing cells, rendering themincreasingly dependent on BCL-2 function.Vec, vector; ut, untreated.
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now be primed with death signals requiring sequestration. This iswhat we found by BH3 profiling for both types of mutants (Fig. 2).Therefore, the experiments with ABT-737 and BH3 profiling agreethat erlotinib treatment induces death signaling that converges onthe mitochondrion and causes cell death via the intrinsic apoptoticpathway. This death signaling can be interrupted by mitochondrialBCL-2, keeping the cell alive, but causing priming of BCL-2 withdeath signals, and rendering it newly dependent on BCL-2 functionfor survival.
It is of obvious interest to determine which of the prodeath BH3-only proteins are dynamic participants in the death signaling aftererlotinib’s inhibition of EGFR. We first examined how abundance oflikely candidate proteins varied with time after erlotinib treatment.We performed this assay using BCL-2–protected cells as thisafforded us the possibility of examining death signaling withoutencountering the artifactual protein degradation encountered ifcells are allowed to commit to PCD. The most striking increase wefound was in the levels of BIM (Fig. 3A). Other proteins investigatedshowedmoremodest increases (PUMA, BAX, and BAK). Also notablewas amodest reduction in levels of the antiapoptotic MCL-1 protein.
Because BCL-2 maintains survival by binding the prodeath-signaling molecules upstream of activation of BAX and BAK, it canalso be considered for experimental purposes as a type of ‘‘bait’’used to capture these death-signaling molecules. We would expectmolecules important in death signaling to therefore be found incomplex with BCL-2 after erlotinib treatment. Thus, we examinedwhat prodeath BH3-only proteins were found in coimmunopreci-pitation complexes with BCL-2. We found that both BIM andPUMA could be found at increasing levels in complex with BCL-2after erlotinib signaling (Fig. 3B). Therefore, BIM and PUMA werecandidates for signaling molecules downstream of EGFR inhibitionthat trigger apoptosis.
Currently, the key clinical application of EGFR inhibition incancer is in NSCLC. Therefore, we next turned to NSCLC celllines to study EGFR inhibition in a more clinically relevantmodel. We selected four NSCLC cell lines: PC9, which harbors adelE746_A750 activating mutation in EGFR; HCC827, whichharbors a delE746_A750 activating mutation in EGFR; H3255,which harbors a L858R activating mutation in EGFR; H1650,which harbors a delE746_A750 activating mutation in EGFR; andH1975, which bears the activating L858R/T790M mutations inEGFR. Of note, the T790M mutation confers resistance toerlotinib by steric hindrance. PC9 and HCC827 are sensitive toerlotinib treatment. H1650, despite harboring an activatingmutation in EGFR that is expected to foster erlotinib sensitivity,is resistant to erlotinib, as is H1975 (refs. 7, 8, 34; Fig. 4A). Wenext investigated the temporal pattern of BCL-2 family proteinlevels after erlotinib treatment in four of these cell lines. At3 h after treatment, BIM levels increased strikingly in thetwo sensitive cell lines after erlotinib treatment, but much lessso in the resistant cells (Fig. 4B). Also notable was a subtle butreproducible shift of BIM to a more rapidly migratingform, likely indicative of a change in a posttranslationalmodification. We examined a wider range of proteins at 24 hafter treatment and found a similar pattern (Fig. 4C ; see alsoSupplementary Figs. S1–4). Densitometric analysis suggests thathigher basal BIM levels and posttreatment BIM levels correspondto erlotinib sensitivity (Fig. 4B and C). Of note, subtle PUMA up-regulation seems to correlate with sensitivity to erlotinib, but itsincrease is of lesser magnitude, and less consistent in repeatedexperiments (Supplementary Fig. S5; data not shown). Levels of
MCL-1 decrease in HCC827 but not other cell lines. It is notclear that reductions in MCL-1 reflect changes upstream of theinitiation of apoptosis, however. BAX and BAK levels do notconsistently change with erlotinib treatment (SupplementaryFigs. S1–4).
H1650 offers a particularly interesting example of erlotinibresistance because it bears a mutation that would be expected toallow drug interaction with EGFR. To verify that erlotinib indeedcan target the EGFR in H1650, we examined phosphorylation oftargets downstream of EGFR. These targets became hypophos-phorylated after erlotinib treatment, demonstrating that EGFR
Figure 3. Proapoptotic BH3-only proteins BIM and PUMA are up-regulated byerlotinib and sequestered by BCL2. A, protein lysates were prepared from L858RBA/F3 bcl-2 cells treated with 0.5 Amol/L erlotinib over a time course andseparated by denaturing electrophoresis. Membranes were blotted withantibodies against BCL-2 family proteins as indicated. B, same protein lysates asA were subjected to immunoprecipitation with an antibody specific for humanBCL-2 (6C8 ). Immunoprecipitants were immunoblotted for BIM, PUMA, andBCL-2 proteins [anti–BCL-2 antibody (/100) was used for immunoblotting afterimmunoprecipitation].
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activity is indeed inhibited (Fig. 4D). Notably, because Akt becomeshypophosphorylated, it rules out resistance due to rescue by METamplification, a resistance mechanism recently described (9).Therefore, we have identified a new mechanism of resistance toerlotinib downstream of EGFR inhibition that does not involveMET but rather blocks BIM up-regulation.
BIM up-regulation was the most striking difference betweensensitive and resistant cell lines. We therefore chose to examinefurther the role of BIM in conveying the death signal downstreamof erlotinib treatment. First, we tested whether NSCLC cellscontaining naturally selected mutants of EGFR used the intrinsicmitochondrial pathway of cell death after erlotinib inhibition, as inour Ba/F3 models. We found that BCL-2 inhibited cell death, again
implicating the requirement of the mitochondrial apoptoticpathway (Fig. 5A). We found that BIM and PUMA could both befound sequestered by BCL-2 after erlotinib treatment (Fig. 5B).Again, the up-regulation of BIM was quantitatively more strikingthan that of PUMA, however. In addition, we found that caspase 9activation, a hallmark of mitochondrial apoptosis, accompaniederlotinib killing, further supporting the importance of the intrinsicmitochondrial pathway (Fig. 5C).
If BIM plays a critical role in the communication of deathsignals, then its removal should inhibit death. To test theimportance of BIM, we used siRNA to knock down BIM levels(Fig. 5D). Reduction of BIM levels indeed caused a reducedsensitivity to treatment with erlotinib, consistent with its role as
Figure 4. Proapoptotic BH3-only proteinBIM is markedly increased by erlotinib insensitive cell lines (PC9 and HCC827) butnot in resistant ones (H1650 and H1975 ).A, NSCLC cells (PC9, HCC827, H1650,H1975 , and H3255 ) were treated with theindicated doses of erlotinib. Viability wasmeasured by CCK-8 colorimetric assay(Dojindo). B and C, NSCLC cells (PC9,HCC827, H1650 , and H1975 ) were treatedwith indicated doses of erlotinib for 3 h (B)and 24 h (C) and harvested for Westernblot analysis for BCL-2 family proteins.Actin is a loading control. BIM-EL/Actinratio for the 3-h blotting was obtained afterperforming densitometric analysis on therespective blot. BIM-EL/Actin ratio for the24-h blotting was shown as a mean of twoindependent blotting (left ; data not shown).D, cell line H1650 was treated with theindicated concentrations of erlotinib. Wholecell lysates were evaluated for proteinlevels and phosphorylation status ofEGFR, AKT, and Erk as indicated byimmunoblot. C, DMSO-treated controlcells.
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a key signaling molecule downstream of activated EGFR activityin NSCLC (Fig. 5D). Similar reduction of PUMA levels had nodetectable effect on erlotinib sensitivity, suggesting a morelimited role for this protein in erlotinib-induced death signaling(Supplementary Fig. S5).
Discussion
Epithelial cancers are responsible for the vast majority ofmorbidity and mortality caused by cancer. An exciting newstrategy has focused on the targeting of activated EGFR moleculesthat are often present in carcinomas. This strategy has reachedclinical fruition in NSCLC with the approval of erlotinib for thesecond- or third-line treatment of NSCLC. Erlotinib has beenshown to induce responses in cancers that bear activatingmutations in EGFR. The molecular pathways connecting EGFRinhibition to cell death have been poorly understood, however.
Furthermore, the alterations in signaling pathways that result inresistance are still incompletely understood.
Here, we definitively connect inhibition of activated EGFR byerlotinib with the intrinsic, or mitochondrial, cell death pathwaycontrolled by the BCL-2 family of proteins. BCL-2, which protectsagainst mitochondrial cell death, inhibits erlotinib toxicity.Moreover, mitochondria isolated from erlotinib-treated, BCL-2–protected cells expressing activated EGFR show evidence ofbearing significant quantities of death signals destined for themitochondrial pathway, as shown by BH3 profiling. We showedthe participation of the mitochondrial pathway both in a modelhematopoietic cell line as well as in NSCLC lines, suggesting thatthis is a general feature to be expected of inhibition of activatedEGFR in many different cellular contexts.
We identify BIM induction as a key step in signaling EGFRinhibition to the intrinsic apoptotic pathway. We, furthermore,show that reduction of BIM by RNAi affords protection against
Figure 5. Sequestering BIM via BCL-2overexpression or decreasing BIM proteinlevels protects cells from death triggeredby erlotinib. A, Western blot analysis ofFlagBCL-2 expression level in transfectedHCC827 cells and cell viability [determinedby Annexin V flow cytometry analysis(FACS )] of parental and transfectantHCC827 cells treated with erlotinib for48 h. Results of cell viability were shownas a percentage of control cells; error bars,SD of triplicate experiments. Vec, vector.B, protein lysates prepared from BCL-2expressing HCC827 cells (control ortreated with erlotinib) were used forimmunoprecipitation (IP ) with an antibodyspecific for human BCL-2 (6C8). Bothprotein lysates and immunoprecipitateswere separated by electrophoresis andimmunoblotted for BIM, PUMA, and BCL-2[anti–BCL-2antibody (/100) was used forimmunoblotting after immunoprecipitation].C1 and C2 were duplicates of DMSOtreated samples. *, a nonspecificcrossreacting band. C, caspase 9 activityassay. BCL-2 or vector expressingHCC827cells were treated with erlotinib(0.1 Amol/L) for 6, 24, and 48 h. At eachtime point, cells were lysed and analyzedfor caspase 9 activity using ApoAlertProfiling Assay plate. The fold changes(erlotinib-treated versus control cells) wereshown from two independent experiments.Columns, mean; bars, SD. E, erlotinib.D, BIM levels were analyzed byimmunoblot in PC9 cells transfected witheither scrambled or BIM siRNA and treatedwith erlotinib. Mock, scramble, or BIMSiRNA–transfected PC9 cells were alsosubjected to viability assay by Annexin Vflow cytometry analysis. Viability wasshown as a percentage of control cells;error bars, SD of triplicate experiments.
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erlotinib-induced apoptosis, supporting a causal role for BIM inthis death signaling. Our comparison of NSCLC cell lines alsosupports a role for BIM in death signaling. It is striking inFig. 4B and C that BIM levels are markedly increased in the celllines sensitive to erlotinib (PC9 and HCC827) but not in thoseresistant to erlotinib (H1650 and H1975).
Although we have shown that BIM plays an important role indeath signaling, this does not rule out the participation of otherBCL-2 family proteins. For instance, we note that the proapoptoticBH3-only protein PUMA is up-regulated in cells sensitive toerlotinib. This is interesting, as PUMA was initially identified as aprodeath protein transcriptionally activated by p53. Subsequently,PUMA up-regulation has been observed under conditions thatseem to be p53 independent. Because erlotinib is not genotoxic,PUMA up-regulation in this case is likely also to be p53 inde-pendent. However, our knockdown results suggest that PUMA playsa more limited role than BIM in death signaling after erlotinibtreatment. In addition, we cannot rule out contribution by one ofthe many other BH3-only proteins that were not examined.However, the BIM siRNA results suggest that a substantialproportion of the BH3-only death signaling is performed by BIM.The balance can be tipped in favor of apoptosis by loss ofantiapoptotic proteins, too. MCL-1 is one of the more dynamicantideath proteins. Although we found MCL-1 levels to decrease
after erlotinib treatment in Ba/F3 cells (Fig. 3A), a similar decreasewas not consistently found in erlotinib-treated NSCLC cell linestested (Supplemtary Figs. S1–4). In addition, increases inproapoptotic BAX and BAK were not consistently found inerlotinib-treated NSCLC cells (Supplementary Figs. S1–4).
Although lung cancers bearing activating mutations in EGFRusually respond to erlotinib, the eventual development ofresistance is very common. In many of these cases, the cause ofresistance is at the level of the target. Many resistant clones survivedue to the introduction of secondary mutations in EGFR mutantsthat are poorly targeted by erlotinib due to steric hindrance. Inothers, it has recently been shown that activation of MET cansubstitute for EGFR signaling (9). However, in a significant portion,no change in EGFR sequence or in MET can be identified. Thissuggests that resistance takes place in the death-signaling pathwaysomewhere downstream of drug-reaching target. We show herethat the proteins in the BCL-2 family are excellent candidates forbeing altered to allow resistance. For instance, we show thatincreased BCL-2 overexpression or decreased BIM expression canboth afford protection from erlotinib. It will be interesting to testwhether either of these alterations are selected for in vivo ;subsequent studies on clinical samples will include investigationof levels of these proteins. Excitingly, we find that a BCL-2antagonist, ABT-737, can effectively abrogate resistance to erlotinibcaused by BCL-2 overexpression. Should clinical samples ofpatients resistant to erlotinib show increased BCL-2 expression,addition of a BCL-2 antagonist such as BCL-2 would be an obviousintervention for clinical investigation.
We also identify a novel mechanism of erlotinib resistance in aNSCLC cell. H1650 harbors del E746_A750 EGFR activatingmutation, and erlotinib completely suppresses both EGFR phos-phorylation and its downstream signaling transduction at low dose(Fig. 4D). However, H1650 is not killed by erlotinib treatment,which suggests the existence of resistance mechanisms down-stream of EGFR inhibition. Interestingly, BIM induction upontreatment with erlotinib in H1650 was greatly reduced inmagnitude and significantly delayed compared with other erlotinibsensitive NSCLC cell lines. Therefore, we can place the source ofresistance in H1650 downstream of EGFR activity inhibition butupstream of BIM activation (Fig. 6).
The signaling pathways that mediate up-regulation of BIMafter erlotinib treatment are not yet fully elucidated. BIM levelsand function have been shown to be subject to control at thelevel of transcription, proteosomal degradation, mRNA stability,phosphorylation, caspase cleavage, and subcellular localization(35–43). Some of these mechanisms are in turn controlled byphosphorylation pathways, including MAP/ERK kinase/extracel-lular signal-regulated kinase, p38MAPK, Ras/Raf, and phospha-tidylinositol-3-OH/mammalian target of rapamycin, so thatintegration with EGFR signaling is certainly plausible (44).Clearly, many testable hypotheses of BIM up-regulation areimmediately suggested and are the subject of current investiga-tion. Once one thoroughly understands the pathways at work inBIM up-regulation, one can begin to design rational strategies toaugment the up-regulation of BIM in conjunction with erlotinibtreatment, or to restore it where lost in clones that acquireresistance to erlotinib. It is noteworthy that it seems thatchanges in posttranslational modification occur to BIM afterEGFR inhibition (Fig. 4B and C ; Supplementary Figs. S1 and S2).It has been shown that phosphorylation of BIM decreases itsproapoptotic function or half-life in conjunction with growth
Figure 6. Model of how apoptosis is induced by erlotinib and how it may beblocked. Inhibition of activated EGFR by erlotinib results in BIM up-regulationdownstream of loss of kinase activity. H1975 is resistant based on a mutation thatinhibits the binding of erlotinib to EGFR. H1650 is resistant based on a novelmechanism that blocks BIM up-regulation downstream of kinase inhibition.BCL-2 can block apoptosis by sequestering BIM and other proapoptoticmolecules generated by erlotinib. BCL-2–derived resistance can be reversed byABT-737. PC9 and HCC827 are killed by the mitochondrial, or intrinsic, apoptoticpathway, downstream of BIM activation.
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factor signaling (35, 36, 38–43). An interesting hypothesis worthyof further study is that loss of EGFR signaling induces loss ofBIM phosphorylation, resulting in an augmentation of theproapoptotic signal.
Here we present results that clarify the death-signaling pathwaysthat are used after erlotinib’s inhibition of activated EGFR incancer. Understanding the details of death pathways used bytargeted therapies is essential in understanding how to predictwhat cancers will respond and what rational combinationtherapies might be most efficacious. Furthermore, understandingthe death pathways assists in the understanding of inducedresistance and in the design of strategies to counteract resistance.Our knowledge of the molecular operation of cell death pathwayshas increased tremendously in the past decade. We are now at the
stage where this knowledge can begin to be put to use for theimprovement of cancer therapy.
Acknowledgments
Received 5/29/2007; revised 9/4/2007; accepted 10/9/2007.Grant support: Lee Foundation Fellowship (J. Deng); NIH grant K08 CA10254
(A. Letai); Dana-Farber/Harvard Cancer Center Specialized Program of ResearchExcellence in Lung Cancer NIH grant P20 CA90578 (T. Shimamura and G. Shapiro);NIH grant R01 CA90687 (G. Shapiro); and Cecily and Robert Harris Foundation(K-K. Wong).
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Saul Rosenberg of Abbott Laboratories for providing ABT-737 andenantiomer and Henry Haringsma for technical assistance. A. Letai is a founder ofEutropics Pharmaceuticals and a member of its scientific advisory board.
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Erlotinib Uses BIM to Kill via Mitochondrial Pathway
www.aacrjournals.org 11875 Cancer Res 2007; 67: (24). December 15, 2007
2007;67:11867-11875. Cancer Res Jing Deng, Takeshi Shimamura, Samanthi Perera, et al. Inhibition to the MitochondrionDeath Signaling from Epidermal Growth Factor Receptor Proapoptotic BH3-Only BCL-2 Family Protein BIM Connects
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