BACKGROUND
the erbb family receptors belong to the receptor tyrosine kinases  and consist of four members; erbb <dig> , erbb <dig>  erbb <dig> and erbb <dig>  <cit> .
egfr is distributed various tissues of the human body  <cit> , and plays a critical role in the regulation of a variety of cellular responses ranging from cell differentiation, growth, proliferation, apoptosis, migration and adhesion  <cit> .
egfr is frequently overexpressed in various human tumors including non-small-cell lung cancer  and is associated with poor outcome  <cit> .
in many cases, enhanced egfr signaling leads to abnormal cellular processes and often induces cancer  <cit> .
certain nsclc patients have mutations at specific amino acid residues in the kinase domain of egfr and show altered responsiveness to gefitinib , an egfr tyrosine kinase inhibitor.
the l858r substitution  is one of the most frequently reported mutations  <cit>  and shows good responses to gefitinib  <cit> .
it was reported that the l858r mutation enhances gefitinib sensitivity due to a structural change in the kinase domain resulting in an increased binding affinity of gefitinib for its atp binding pocket in vitro  <cit> .
on the other hand, a large scale binding assay using different types of kinases showed that the difference in binding affinity of the egfr itself may not have a great effect on gefitinib sensitivity  <cit> .
based on these observations, we speculated that other unknown factors affect gefitinib sensitivity in vivo rather than alteration of the binding affinity.
so far, cells with the l858r-mutated egfr have been reported to have two characteristics.
first, mig <dig>  is highly expressed in the l858r-mutated egfr cells  <cit> .
mig <dig> is an adaptor molecule that binds to an activating kinase domain of an egfr  <cit>  and functions as a negative regulator of egfr kinase  <cit> .
mutation and downregulation of mig <dig> are often observed in human lung cancer cell lines  <cit>  and also correlate with a reduced survival rate in breast cancer patients  <cit> .
secondly, ubiquitin-dependent egfr degradation mediated by cbl is enhanced in the l858r cells  <cit> .
both of these two characteristics seem to contribute to the negative regulation of the egfr signaling pathway.
however, no mechanistic explanation has been found for the contributions of these molecules to the gefitinib sensitivity of the l858r mutation.
recent studies showed that dynamics and regulation of the intracellular signaling cascades are efficiently elucidated with an assistance of computational simulations  <cit> .
to obtain a logical understanding of the gefitinib sensitivity associated with l858r mutation, the mathematical analysis of the egfr signaling pathway should be more preferable rather than sole experimental representations.
in this study, we used experimental and computational approaches to investigate regulatory mechanisms that distinguish cell-specific gefitinib sensitivity in h <dig> human nsclc cell lines.
we have modified the existing kinetic model of the egfr signaling pathway and built new models for h <dig> wild type , h <dig> with overexpressed wild type egfr , and h <dig> overexpressing the egfr with l858r mutation .
the three types of cells showed different signaling dynamics in response to egf stimulation.
overexpression of wild type egfr induced high and sustained phosphorylation of egfr, shc, mek  and erk , while the l858r mutation reduced these response levels.
in addition, h1299l858r, which is supposed to be more sensitive to gefitinib than h1299egfr-wt, was effectively inhibited by gefitinib administration at the downstream part of the signaling pathway  compared with h1299egfr-wt, but, surprisingly, not at the upstream part of the pathway .
the model incorporated mig <dig>  but not cbl overexpression, successfully captured the signaling behavior observed in our experimental data, implying that mig <dig> is responsible for enhancing gefitinib sensitivity.
detailed computational analyses revealed that mig <dig> amplifies gefitinib sensitivity at the steps of mek phosphorylation/dephosphorylation and erk phosphorylation/dephosphorylation.
we experimentally verified that overexpression of mig <dig> contributed to suppressing cellular growth in the presence of gefitinib.
our analysis further suggested that the combination of mig <dig> and gefitinib exhibits a synergistic effect in inhibiting egfr signaling pathway.
methods
cell culture
h <dig> human lung cancer derivatives, h1299wt, h1299egfr-wt and h1299l858r, were established as described elsewhere  <cit> .
cells were maintained in rpmi <dig> medium supplemented with 10% fetal bovine serum and  <dig> mm sodium pyruvate.
prior to growth hormone treatment, the cells were serum-starved for 16- <dig> hours.
for the egfr kinase inhibition, gefitinib  was added  <dig> minutes prior to the growth factor treatment.
the cells were incubated with  <dig> nm of egf for  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> minutes and then washed two times with phosphate buffered saline  and lysed with bio-plex lysis buffer .
cell lysates were cleared by centrifugation, and the total protein concentration of the supernatant was determined using a protein assay reagent  and analyzed by western blot.
cells that were not treated with growth hormone were used as the control.
western blot analysis
sds-page and membrane transfer were performed using standard protocols.
antibodies against anti-phospho-egfr , doubly phosphorylated p44/ <dig> erk , erk, phospho-mek1/ <dig> , mek, mig <dig> and actin were purchased from cell signaling technology, inc. .
anti-phospho-shc , anti-shc antibodies and anti-egfr antibodies were purchased from upstate biotechnology .
protein band intensities were quantified using a densitometer .
normalization procedure is described in earlier study  <cit> .
briefly, the maximum value of protein phosphorylation level among three cell lines  was set to  <dig> and the values at t =  <dig> minutes were set to  <dig> under the assumption that all the proteins were inactive before egf stimulation.
we considered that total protein level of egfr is equal in egfr-wt and l858r cells.
all concentrations of shc, mek and erk were considered to be equal in three cell lines .
mig <dig> overexpression
the mig <dig> gene was amplified from a human errfi <dig> cdna purchased from origene  using the primers; mig <dig> forward 5' gct tgt cga ctc tag aga tgt ccc aga ata agg cac aat g-3' and mig <dig> reverse 5'-gcg gcc gca atc tag atc tgc tga acc atg acc cca ag-3'.
the resulting dna fragment was cloned into the vector pcmv-6-neo  using the xba i restriction site.
cells were seeded in  <dig> well plates at  <dig> ×  <dig> cells/well.
transfection of the mig <dig> gene was performed using the lipofectamine ltx  and combimag magnetofection kit  according to manufacturer's protocol.
control cells were transfected with pcmv-6-neo vector.
after  <dig> hours of transfection, cells were supplemented with serum free rpmi <dig> media.
the following day, cells were treated with  <dig> nm egf in the presence or absence of gefitinib.
cell viability assay
cell viabilities of h <dig> cells were measured by an mtt  cell proliferation assay  <dig> days after stimulation with or without  <dig> nm egf in the presence of various doses of gefitinib  using the cell count kit sf .
the cell viability was determined by optical density  at  <dig> nm.
computation
to model egfr signaling network, we adopted a deterministic ordinary differential equation  model.
model scheme is described in additional file  <dig>  table s1- <dig>  additional file  <dig>  table s <dig> and  <dig> summarize the biochemical reactions with  <dig> components and  <dig> differential equations, which are given by mass action or michaelis-menten kinetics.
additional file  <dig>  table s <dig> and  <dig> list the parameter values and the initial concentrations of the cellular signaling molecules.
these values were estimated based on the parameter ranges which were listed in additional file  <dig>  table s <dig>  our pathway network is drawn by cell designer  <dig>  which is an systems biology markup language - compliant application, and is available with this publication .
the parameter estimation problem is defined as a function optimization problem to minimize the sum of the squared error:
err=∑cell type∈{wt, egfr-wt, l858r models}∑protein∈p∑datapoint∈d{experimental data–simulated value} <dig>
where the sets p = {phosphorylated egfr, shc, mek, erk} and d = { <dig>   <dig>   <dig>   <dig> minutes after egf stimulation} are an experimentally observed values .
we used a technique to decompose the parameter estimation problem of our model into two subproblems, reaction steps from  <dig> to  <dig>  and from  <dig> to  <dig> .
the problem decomposition technique is an effective means to resolve the high-dimensionality and can only be applied to enzymatic reactions given by michaelis-menten kinetics.
since the upstream region affect the downstream subproblem, the subproblems were solved in order, from the upstream  to the downstream .
as a parameter estimator, we used the genetic algorithm with genetic local search with distance independent diversity control  by extending the basic idea of a genetic algorithm with distance independent diversity control  to coarse grained parallelization  <cit> .
the glsdc program was executed on the the riken integrated cluster of clusters  system.
the model was implemented with matlab r2008a , and ''ode15s'' function was applied to solve the odes .
"ode15s" function is a variable order solver based on the numerical differentiation formulas  and is a multistep solver.
the function is used when the problem is a differential-algebraic or stiff equation .
additive, antagonistic, or synergistic effect classification analysis
classification of additive, antagonistic, or synergistic effect is determined by comparing the response to a combinatorial perturbation with that to a single perturbation  <cit> .
this classification analysis has been mainly used to categorize the efficacy of combinatorial drugs into three types, considering drug dose as a perturbation.
in the present analysis, we made some modifications not to lose the original meaning so that we could categorize the erkpp inhibitory effect by the combinatorial perturbations of mig <dig> effect and gefitinib.
as an index for the efficacy of perturbation, we used the concentration of erkpp at t =  <dig> minutes.
the perturbation for the administration of gefitinib was expressed by varying the parameter k <dig> .
the perturbation for the effect of mig <dig> was expressed by varying both k <dig>  and k <dig> , because only these two parameters contribute to the strong inhibition of erkpp, although we assumed that four parameters  were affected by mig <dig> in l858r model a. given the values of k <dig> or k <dig> that individually achieve x/2% erkpp inhibition, the value of paired perturbation additively produces x% erkpp inhibition.
therefore, the combinatorial effect at an inhibitory intensity x is categorized as additive, antagonistic, or synergistic according to whether the paired perturbation produces erkpp inhibition equal to, less than, or more than x%.
for example, the combination of the value of k <dig> and k <dig> which result in 10% inhibition of erkpp individually is categorized into additive, antagonistic, or synergistic effect, according to whether the inhibition level in erkpp is equal to, less than, or more than 20%.
RESULTS
mathematical model of egfr-erk signaling in h <dig> lung cancer cells
to evaluate the dynamics of the signal transmission from egfr to its downstream elements, we used the network model introduced by kholodenko et al  <cit> , hatakeyama et al.
<cit> , and wolf et al  <cit>  with some modifications.
figure  <dig> shows the egfr signaling pathway considered in the current study.
the pathway starts from the egfr located in the cell membrane and is composed of  <dig> reaction steps.
egf first binds to the egfr and causes receptor dimerization, then receptor autophosphorylation occurs at particular tyrosine residues in the cytoplasmic domains   <cit> .
phosphorylated egfr is dephosphorylated by protein phosphotyrosine phosphatases   <cit> .
some of the phosphorylated dimers are internalized by binding of cbl and subsequently degraded   <cit> .
this receptor degradation is one of the most important processes for preventing over-signaling.
other phophorylated dimers associate with the grb <dig> /sos  complex via shc    <cit> .
this complex can dissociate, yielding the egfr dimer, the grb2/sos complex, and shc .
after recruiting grb2/sos with the phosphorylated egfr dimer to the plasma membrane, sos activates ras by exchanging gdp for gtp   <cit> .
in an opposing reaction, deactivation of ras is accelerated by gaps  associated with egfr .
binding of gap to the phosphorylated egfr is a key step to control the output of the signaling pathway  <cit> .
egf stimulation induces recruitment of gap to the membrane  <cit> , and gap is strongly activated after binding to the phosphorylated egfr.
ras deactivation normally keeps its gtpase activity low.
the gtp-bound ras can then translocates raf <dig> to the cell membrane for its activation   <cit> , which is also reversible .
activated raf <dig> activates mek by phosphorylation of two serine residues , and the activated mek phosphorylates erk on threonine and tyrosine residues   <cit> .
the mapk cascade is negatively regulated by pp2a  for the dephosphorylation of mek  and by mkp <dig>  for the dephosphorylation of erk   <cit> .
after its translocation to the nucleus, activated erk regulates gene expression by phosphorylating transcription factors such as elk and myc  <cit> .
to investigate cell-specific egfr signaling dynamics, we constructed an h1299wt model , an h1299egfr-wt model , and two alternate h1299l858r models .
the differences among these models are summarized in table  <dig>  to simulate the effect of egfr overexpression in h1299egfr-wt and h1299l858r cells, the initial concentrations of egfr in the respective models were assumed to be higher than that in the wt model.
also, we constructed two l858r models based on h1299l858r cell-specific characteristics.
in the first model , mig <dig> was added to the egfr-wt model because mig <dig> is highly endogenously expressed in h1299l858r cells  <cit> .
for simplicity, the effect of mig <dig> was not described explicitly, but was realized by modifying four parameters in the egfr-wt model.
the modified parameters were the rate constant for the forward reaction of the egfr phosphorylation , the binding of egfr to cbl , gap , and shc  .
these modifications were included to mimic the effect of mig <dig> overexpression, which leads to suppression of egfr phosphorylation and binding of the egfr dimer to other proteins  <cit> .
in the second model , the initial concentration of cbl was increased compared to that in the egfr-wt model, because h1299l858r cells showed an increase in ubiquitination compared to h1299egfr-wt cells  <cit>  and receptors in cbl overexpressing cells underwent more rapid ligand-induced ubiquitination compared to control cells  <cit> .
the values of other parameters were the same as those in the egfr-wt model.
time-course and dose-dependent phosphorylation by egf stimulation
first, we used experimental and computational approaches to investigate the time-course of egfr signaling dynamics.
figure  <dig> and additional file  <dig>  figure s1a show the experimental results.
the graphs show the time-courses of phosphorylation levels after stimulation with  <dig> ,  <dig>  and  <dig> nm egf measured for four key proteins: egfr, shc, mek and erk.
h1299egfr-wt cells showed the highest level of phosphorylation in all four proteins, whereas the h1299wt cells showed the lowest and the h1299l858r cells were intermediate.
overexpression of egfr induced sustained and strong signaling activity, while the l858r mutation reduced signaling particularly in the upstream part of the signaling pathway .
in contrast, the differences in the time-course kinetics among the three derivatives became less obvious in the downstream part of the signaling pathway .
the simulation results of wt, egfr-wt, and two l858r models for egf stimulation  are shown in figure  <dig> and additional file  <dig>  figure s <dig>  as described in the previous section, most of the parameters were common among the four models.
despite using these common parameters, the models successfully captured the variations in time-course activation dynamics, and the simulation results were fairly consistent with the experimental results for all three h <dig> cell derivatives .
next, the egf ligand-dose response was examined to investigate how the l858r mutation affects cooperativity of egfr signaling.
figure  <dig> and additional file  <dig>  figure s1b show the experimental results in the phosphorylation levels of egfr, shc, mek, and erk in the egfr pathway when the dose of egf was varied.
the phosphorylation levels of egfr and shc gradually increased as a function of the egf concentration in both h1299egfr-wt and h1299l858r cells, while mek and erk were highly phosphorylated even at egf concentrations as low as  <dig> nm.
the phosphorylation of egfr and shc in the h1299l858r cells showed a smaller dynamic range compared to that of the h1299egfr-wt cells, whereas there was no significant difference between the two cell types in mek and erk phosphorylation patterns.
again, the l858r mutation reduced the phosphorylation levels in the upstream part of the signaling pathway  in an egf-dose dependent manner, but had little effect on the downstream part .
the simulation results imply that the models fairly reproduced the ligand-dose dependent behavior of the h1299egfr-wt and h1299l858r cells .
also, both l858r models a and b seem to equally reproduce the time-course and the egf dose-dependency obtained with the h1299l858r cells.
additional analyses are required to distinguish the model that represents the behaviors of the signaling dynamics associated with l858r mutation.
mig <dig> plays a role in transmitting the effect of gefitinib to the downstream part of the egfr signaling pathway
to clarify unknown other factors that affect gefitinib sensitivity, we investigated the gefitinib-dose response effect on the egfr signaling pathway.
figure 4a and additional file  <dig>  figure s1c show the experimental results of phosphorylation of the signaling proteins in the presence of different doses of gefitinib.
the h1299egfr-wt and h1299l858r cells showed similar dose-dependent responses in the upstream proteins egfr and shc.
however, the h1299l858r cells were more sensitive to gefitinib administration at the phosphorylation of the downstream proteins, mek and especially erk, compared to the h1299egfr-wt cells, which were essentially insensitive to gefitinib for erk phosphorylation .
figure 4b shows the simulation results of the gefitinib dose-response.
the effect of the gefitinib was mimiced by changing the kinetic parameter k <dig> of egfr phosphorylation.
α indicates the multiplying coefficient for k <dig>  the results indicate that the l858r model a, including mig <dig>  successfully reproduced the inhibitory effect for erk at higher concentrations of gefitinib, whereas model b, which included the effect of cbl, failed to reproduce this response.
we confirmed that l858r model a with other parameter sets that have similar cost function values yields the same trends with regard to the role of mig <dig> in gefitinib sensitivity .
also, we found that egfr-wt model at lower concentrations of egf is as sensitive to gefitinib for erk phosphorylation as l858r model a .
based on these results, model b is inappropriate as the mechanism to explain the gefitinib sensitivity, whereas model a remains as a viable candidate.
although the sensitivity to inhibition of phosphorylation by gefitinib was different in the downstream proteins  between h1299egfr-wt and h1299l858r cells, the difference was small in the upstream proteins .
therefore, it seemed likely that particular reaction steps in the pathway would amplify the small difference observed in the upstream proteins .
to identify the critical steps, we analyzed the contribution of each parameter to the downstream phosphorylation influenced by gefitinib.
we used an index that indicates the ratios of the upstream signaling activity to the downstream signaling activity in the presence of gefitinib.
the output of the upstream signaling was the total phosphorylated shc, and the downstream output was the total phosphorylated erk.
when rx is defined as the ratios of total phosphorylated x with gefitinib to total phosphorylated x without gefitinib, the index rshc/rerk was calculated for every combination of the four parameters  affected by mig <dig>  as a result, the combination of a lower k <dig>  and k <dig>  contributed to effectively inhibit the total phosphorylation of erk in the presence of gefitinib .
the values in each panel indicate rshc/rerk calculated by using the values of parameters in l858r model a for two changing parameters and those in egfr-wt model for two unchanging parameters.
the value of rshc/rerk for l858r model a is  <dig> .
next, we analyzed which downstream reactions were influenced by the combination of these two parameters.
in this case, e11p/shcp/grb2/sos was used as the upstream output, and the downstream outputs were rasgtp, raf1a, mekp, mekpp, erkp, and erkpp.
when ry is defined as the ratios of y with gefitinib to y without gefitinib, figure 5b shows re11p/shcp/grb2/sos/ry by varying the values of k <dig> and k <dig> .
the values in each panel indicate re11p/shcp/grb2/sos/ry calculated by using the values of k <dig> and k <dig> in l858r model a and k <dig> and k <dig> in egfr-wt model.
based on this analysis, we found high gefitinib sensitivity in mekpp, erkp, and erkpp.
these results indicate that the steps of mek phosphorylation/dephosphorylation  and erk phosphorylation/dephosphorylation  amplify the gefitinib sensitivity.
the combination of mig <dig> and gefitinib has a synergistic effect in inhibiting egfr signaling
the only difference between the egfr-wt model and the l858r model a is in the negative egfr regulation produced by mig <dig>  therefore, which can be experimentally verified by overexpressing mig <dig>  we have known that expression level of mig <dig> is reversely correlated with erk phosphorylation level in the h <dig> derivatives with various egfr mutations  <cit> .
to study its effect on the upstream signaling, we performed western blotting for phosphorylated egfr.
figure 6a shows that mig <dig> overexpression more inhibited egfr phosphorylation in the presence of gefitinib.
therefore the effect of mig <dig> for gefitinib administration was further studied using mtt cell proliferation assay.
at a high concentration  of gefitinib, cell growth was suppressed in the cells with mig <dig> overexpression, but not in the h1299egfr-wt cells .
this result indicates that mig <dig> indeed enhances the inhibitory effect of gefitinib as our mathematical model had predicted.
we next analyzed the effects of a combinatorial perturbation of mig <dig> and gefitinib on the signaling inhibition.
combinations of perturbations can be categorized into three interaction types: additive, antagonistic, or synergistic, according to whether the combination of two perturbations produces an effect equal to, less than, or larger than that expected based on the individual effects of the single perturbations  <cit> .
we analyzed the effect of the combination of mig <dig> and gefitinib on erkpp  and found that the combination of mig <dig> and gefitinib exhibits a synergistic effect .
this result indicates that synergism is produced by the dual inhibition of the step of egfr phosphorylation  which both mig <dig> and gefitinib inhibit and the step of binding of egfr to shc  which mig <dig> alone inhibits.
discussion
overexpression or mutation of egfr has been observed in lung cancers  <cit> , and these molecular changes affect the prognosis and treatment sensitivity of patients  <cit> .
those abnormalities could cause changes in overall titers of signaling networks at the molecular, the cellular, and even the individual levels.
mathematical model is helpful for understanding of the mechanical aspects of interconnected signaling network and predicting input-output behaviors in the pathway.
however, it is often very difficult to explain the variation of reactants in signal transduction pathway using a single unified mathematical model.
in this paper, we attempted to build such a unified model that could uncover the cell-specific regulatory mechanisms produced by overexpression and mutation of the egfr and the association with gefitinib sensitivity.
the model used in this paper successfully reproduced the experimental observations concerning the activation of the key proteins in the pathway and discriminated the roles of mig <dig> and cbl in gefitinib sensitivity.
the model was based on kinetic equations, and most of the parameters for these equations were common for all models.
the differences in the parameters were confined to specific steps and proteins - egfr overexpression, inhibitory effects caused by mig <dig>  and cbl overexpression leading to the degradation of egfr.
our results revealed that the effectiveness of gefitinib in cells is largely affected by not only on its direct binding affinity with egfr but also on the presence of an additional molecule, mig <dig>  according to recent reports, the sensitivity to kinase inhibition reflects intrinsic differences in the binding affinity of the egfr mutants such as l858r, g719s, and exon <dig> deletions  <cit> .
yun et al  <cit>  showed that gefitinib directly binds more tightly to the l858r mutant than to the wild type egfr in vitro, while fabian et al  <cit>  indicated that egfr with gefitinib sensitive mutations does not differ from wild type egfr in terms of gefitinib binding affinity.
this would suggest that the stronger interaction of the mutated egfrs with gefitinib may not be the only one mechanism for the good clinical response to gefitinib in nsclc  <cit> .
this inconsistency in previous reports suggested to us that unknown factors may play an important role in gefitinib sensitivity.
the gefitinib-dose response study  showed that the difference in the phosphorylation levels of the downstream proteins  inhibited by gefitinib is large between h1299egfr-wt and h1299l858r cells, whereas the difference in the upstream proteins  is small.
by a detailed computational analysis, we found that mig <dig> has an important role in propagating the gefitinib effect at the steps of mek phosphorylation/dephosphorylation and erk phosphorylation/dephosphorylation.
additionally, the combination of the inhibition of egfr phosphorylation  and the inhibition of binding of egfr to shc  produced a synergistic inhibitory effect on egfr signaling.
therefore, mig <dig> could be one of the critical factors to explain gefitinib sensitivity at cellular level.
we constructed the model by referring to the earlier studies on mig <dig> functions  <cit> .
however, the model could be modified and improved when novel mechanism of mig <dig> in the regulation of egfr or new regulators associated with the egfr l858r mutation are identified by further studies.
our results shown in figures  <dig> and  <dig> are consistent with a previously published report that egfr with the l858r mutation did not have stronger egfr phosphorylation upon egf stimulation when compared to the wild type egfr in h <dig> cells  <cit> .
on the other hand, guha et al  <cit>  and yun et al  <cit>  observed significantly high phosphorylation of the l858r-mutated egfr compared with wild type egfr expressed in hbec cells and sf <dig> cells.
this inconsistency among cell types may be explained by the relative levels of mig <dig>  which is highly expressed when egfr kinase is in an active state.
CONCLUSIONS
overall, the analysis presented in this paper allows understanding of the impacts of cancer-related abnormalities on the egfr signaling pathway.
also, we demonstrate the feasibility of using computational models to predict one of the determinants for the evaluation of drug sensitivities.
despite the fact that a new drug may help prevent the deaths of thousands of patients, there are many instances where the patients become severely ill or die because of serious unwanted side-effects.
hence, in prescribing medications appropriate for individual patients, there is a clear need for guidance in predicting side-effects and drug sensitivity.
it would be no exaggeration to say that the side-effects could not be predicted in advance, since signaling pathways are very complex.
we believe that in part such guidance can be predicted by computational modeling of appropriate signaling pathways.
authors' contributions
yn carried out the mathematical modeling and analysis work, and wrote the manuscript.
tnag helped to draft the manuscript.
ru, ys, and sft carried out the experimental laboratory work.
tnakak and tnaka proposed a basic idea of the mathematical methods.
ht participated in the design and coordination of the study.
all of the work was supervised by mo.
all authors have read and approved the final version of the manuscript.
supplementary material
additional file 1
supplementary information.
this pdf file contains all additional figures and tables referenced in the text.
click here for file
additional file 2
h <dig> model.
this xml file contains our model represented by sbml.
click here for file
acknowledgements
we are grateful to dr yi-robg chen  for providing h <dig> derivative cell lines for using in the present paper.
we also thank prof tadashi masuda, dr kaoru mogushi, and dr takeshi hase for a critical reading of the manuscript and helpful discussions.
all parallel computation was performed by using the riken integrated cluster of clusters  facility.

 the erbb family receptors belong to the receptor tyrosine kinases and consist of four members; erbb, erbb erbb and erbb. egfr is distributed various tissues of the human body, and plays a critical role in the regulation of a variety of cellular responses ranging from cell differentiation, growth, proliferation, apoptosis, migration and adhesion. egfr is frequently overexpressed in various human tumors including non-small-cell lung cancer and is associated with poor outcome. in many cases, enhanced egfr signaling leads to abnormal cellular processes and often induces cancer. certain nsclc patients have mutations at specific amino acid residues in the kinase domain of egfr and show altered responsiveness to gefitinib, an egfr tyrosine kinase inhibitor. the l858r substitution is one of the most frequently reported mutations and shows good responses to gefitinib. it was reported that the l858r mutation enhances gefitinib sensitivity due to a structural change in the kinase domain resulting in an increased binding affinity of gefitinib for its atp binding pocket in vitro. on the other hand, a large scale binding assay using different types of kinases showed that the difference in binding affinity of the egfr itself may not have a great effect on gefitinib sensitivity. based on these observations, we speculated that other unknown factors affect gefitinib sensitivity in vivo rather than alteration of the binding affinity. so far, cells with the l858r-mutated egfr have been reported to have two characteristics. first, mig is highly expressed in the l858r-mutated egfr cells. mig is an adaptor molecule that binds to an activating kinase domain of an egfr and functions as a negative regulator of egfr kinase. mutation and downregulation of mig are often observed in human lung cancer cell lines and also correlate with a reduced survival rate in breast cancer patients. secondly, ubiquitin-dependent egfr degradation mediated by cbl is enhanced in the l858r cells. both of these two characteristics seem to contribute to the negative regulation of the egfr signaling pathway. however, no mechanistic explanation has been found for the contributions of these molecules to the gefitinib sensitivity of the l858r mutation. recent studies showed that dynamics and regulation of the intracellular signaling cascades are efficiently elucidated with an assistance of computational simulations. to obtain a logical understanding of the gefitinib sensitivity associated with l858r mutation, the mathematical analysis of the egfr signaling pathway should be more preferable rather than sole experimental representations. in this study, we used experimental and computational approaches to investigate regulatory mechanisms that distinguish cell-specific gefitinib sensitivity in h human nsclc cell lines. we have modified the existing kinetic model of the egfr signaling pathway and built new models for h wild type, h with overexpressed wild type egfr, and h overexpressing the egfr with l858r mutation. the three types of cells showed different signaling dynamics in response to egf stimulation. overexpression of wild type egfr induced high and sustained phosphorylation of egfr, shc, mek and erk, while the l858r mutation reduced these response levels. in addition, h1299l858r, which is supposed to be more sensitive to gefitinib than h1299egfr-wt, was effectively inhibited by gefitinib administration at the downstream part of the signaling pathway compared with h1299egfr-wt, but, surprisingly, not at the upstream part of the pathway. the model incorporated mig but not cbl overexpression, successfully captured the signaling behavior observed in our experimental data, implying that mig is responsible for enhancing gefitinib sensitivity. detailed computational analyses revealed that mig amplifies gefitinib sensitivity at the steps of mek phosphorylation/dephosphorylation and erk phosphorylation/dephosphorylation. we experimentally verified that overexpression of mig contributed to suppressing cellular growth in the presence of gefitinib. our analysis further suggested that the combination of mig and gefitinib exhibits a synergistic effect in inhibiting egfr signaling pathway. methods cell culture h human lung cancer derivatives, h1299wt, h1299egfr-wt and h1299l858r, were established as described elsewhere. cells were maintained in rpmi medium supplemented with 10% fetal bovine serum and mm sodium pyruvate. prior to growth hormone treatment, the cells were serum-starved for 16- hours. for the egfr kinase inhibition, gefitinib was added minutes prior to the growth factor treatment. the cells were incubated with nm of egf for and minutes and then washed two times with phosphate buffered saline and lysed with bio-plex lysis buffer. cell lysates were cleared by centrifugation, and the total protein concentration of the supernatant was determined using a protein assay reagent and analyzed by western blot. cells that were not treated with growth hormone were used as the control. western blot analysis sds-page and membrane transfer were performed using standard protocols. antibodies against anti-phospho-egfr, doubly phosphorylated p44/ erk, erk, phospho-mek1/, mek, mig and actin were purchased from cell signaling technology, inc.. anti-phospho-shc, anti-shc antibodies and anti-egfr antibodies were purchased from upstate biotechnology. protein band intensities were quantified using a densitometer. normalization procedure is described in earlier study. briefly, the maximum value of protein phosphorylation level among three cell lines was set to and the values at t = minutes were set to under the assumption that all the proteins were inactive before egf stimulation. we considered that total protein level of egfr is equal in egfr-wt and l858r cells. all concentrations of shc, mek and erk were considered to be equal in three cell lines. mig overexpression the mig gene was amplified from a human errfi cdna purchased from origene using the primers; mig forward 5' gct tgt cga ctc tag aga tgt ccc aga ata agg cac aat g-3' and mig reverse 5'-gcg gcc gca atc tag atc tgc tga acc atg acc cca ag-3'. the resulting dna fragment was cloned into the vector pcmv-6-neo using the xba i restriction site. cells were seeded in well plates at × cells/well. transfection of the mig gene was performed using the lipofectamine ltx and combimag magnetofection kit according to manufacturer's protocol. control cells were transfected with pcmv-6-neo vector. after hours of transfection, cells were supplemented with serum free rpmi media. the following day, cells were treated with nm egf in the presence or absence of gefitinib. cell viability assay cell viabilities of h cells were measured by an mtt cell proliferation assay days after stimulation with or without nm egf in the presence of various doses of gefitinib using the cell count kit sf. the cell viability was determined by optical density at nm. computation to model egfr signaling network, we adopted a deterministic ordinary differential equation model. model scheme is described in additional file table s1- additional file table s and summarize the biochemical reactions with components and differential equations, which are given by mass action or michaelis-menten kinetics. additional file table s and list the parameter values and the initial concentrations of the cellular signaling molecules. these values were estimated based on the parameter ranges which were listed in additional file table s our pathway network is drawn by cell designer which is an systems biology markup language - compliant application, and is available with this publication. the parameter estimation problem is defined as a function optimization problem to minimize the sum of the squared error: err=∑cell type∈{wt, egfr-wt, l858r models}∑protein∈p∑datapoint∈d{experimental data–simulated value} where the sets p = {phosphorylated egfr, shc, mek, erk} and d = { minutes after egf stimulation} are an experimentally observed values. we used a technique to decompose the parameter estimation problem of our model into two subproblems, reaction steps from to and from to. the problem decomposition technique is an effective means to resolve the high-dimensionality and can only be applied to enzymatic reactions given by michaelis-menten kinetics. since the upstream region affect the downstream subproblem, the subproblems were solved in order, from the upstream to the downstream. as a parameter estimator, we used the genetic algorithm with genetic local search with distance independent diversity control by extending the basic idea of a genetic algorithm with distance independent diversity control to coarse grained parallelization. the glsdc program was executed on the the riken integrated cluster of clusters system. the model was implemented with matlab r2008a, and''ode15s'' function was applied to solve the odes. "ode15s" function is a variable order solver based on the numerical differentiation formulas and is a multistep solver. the function is used when the problem is a differential-algebraic or stiff equation. additive, antagonistic, or synergistic effect classification analysis classification of additive, antagonistic, or synergistic effect is determined by comparing the response to a combinatorial perturbation with that to a single perturbation. this classification analysis has been mainly used to categorize the efficacy of combinatorial drugs into three types, considering drug dose as a perturbation. in the present analysis, we made some modifications not to lose the original meaning so that we could categorize the erkpp inhibitory effect by the combinatorial perturbations of mig effect and gefitinib. as an index for the efficacy of perturbation, we used the concentration of erkpp at t = minutes. the perturbation for the administration of gefitinib was expressed by varying the parameter k. the perturbation for the effect of mig was expressed by varying both k and k, because only these two parameters contribute to the strong inhibition of erkpp, although we assumed that four parameters were affected by mig in l858r model a. given the values of k or k that individually achieve x/2% erkpp inhibition, the value of paired perturbation additively produces x% erkpp inhibition. therefore, the combinatorial effect at an inhibitory intensity x is categorized as additive, antagonistic, or synergistic according to whether the paired perturbation produces erkpp inhibition equal to, less than, or more than x%. for example, the combination of the value of k and k which result in 10% inhibition of erkpp individually is categorized into additive, antagonistic, or synergistic effect, according to whether the inhibition level in erkpp is equal to, less than, or more than 20%.  mathematical model of egfr-erk signaling in h lung cancer cells to evaluate the dynamics of the signal transmission from egfr to its downstream elements, we used the network model introduced by kholodenko et al, hatakeyama et al., and wolf et al with some modifications. figure shows the egfr signaling pathway considered in the current study. the pathway starts from the egfr located in the cell membrane and is composed of reaction steps. egf first binds to the egfr and causes receptor dimerization, then receptor autophosphorylation occurs at particular tyrosine residues in the cytoplasmic domains. phosphorylated egfr is dephosphorylated by protein phosphotyrosine phosphatases. some of the phosphorylated dimers are internalized by binding of cbl and subsequently degraded. this receptor degradation is one of the most important processes for preventing over-signaling. other phophorylated dimers associate with the grb /sos complex via shc. this complex can dissociate, yielding the egfr dimer, the grb2/sos complex, and shc. after recruiting grb2/sos with the phosphorylated egfr dimer to the plasma membrane, sos activates ras by exchanging gdp for gtp. in an opposing reaction, deactivation of ras is accelerated by gaps associated with egfr. binding of gap to the phosphorylated egfr is a key step to control the output of the signaling pathway. egf stimulation induces recruitment of gap to the membrane, and gap is strongly activated after binding to the phosphorylated egfr. ras deactivation normally keeps its gtpase activity low. the gtp-bound ras can then translocates raf to the cell membrane for its activation, which is also reversible. activated raf activates mek by phosphorylation of two serine residues, and the activated mek phosphorylates erk on threonine and tyrosine residues. the mapk cascade is negatively regulated by pp2a for the dephosphorylation of mek and by mkp for the dephosphorylation of erk. after its translocation to the nucleus, activated erk regulates gene expression by phosphorylating transcription factors such as elk and myc. to investigate cell-specific egfr signaling dynamics, we constructed an h1299wt model, an h1299egfr-wt model, and two alternate h1299l858r models. the differences among these models are summarized in table to simulate the effect of egfr overexpression in h1299egfr-wt and h1299l858r cells, the initial concentrations of egfr in the respective models were assumed to be higher than that in the wt model. also, we constructed two l858r models based on h1299l858r cell-specific characteristics. in the first model, mig was added to the egfr-wt model because mig is highly endogenously expressed in h1299l858r cells. for simplicity, the effect of mig was not described explicitly, but was realized by modifying four parameters in the egfr-wt model. the modified parameters were the rate constant for the forward reaction of the egfr phosphorylation, the binding of egfr to cbl, gap, and shc. these modifications were included to mimic the effect of mig overexpression, which leads to suppression of egfr phosphorylation and binding of the egfr dimer to other proteins. in the second model, the initial concentration of cbl was increased compared to that in the egfr-wt model, because h1299l858r cells showed an increase in ubiquitination compared to h1299egfr-wt cells and receptors in cbl overexpressing cells underwent more rapid ligand-induced ubiquitination compared to control cells. the values of other parameters were the same as those in the egfr-wt model. time-course and dose-dependent phosphorylation by egf stimulation first, we used experimental and computational approaches to investigate the time-course of egfr signaling dynamics. figure and additional file figure s1a show the experimental results. the graphs show the time-courses of phosphorylation levels after stimulation with, and nm egf measured for four key proteins: egfr, shc, mek and erk. h1299egfr-wt cells showed the highest level of phosphorylation in all four proteins, whereas the h1299wt cells showed the lowest and the h1299l858r cells were intermediate. overexpression of egfr induced sustained and strong signaling activity, while the l858r mutation reduced signaling particularly in the upstream part of the signaling pathway. in contrast, the differences in the time-course kinetics among the three derivatives became less obvious in the downstream part of the signaling pathway. the simulation results of wt, egfr-wt, and two l858r models for egf stimulation are shown in figure and additional file figure s as described in the previous section, most of the parameters were common among the four models. despite using these common parameters, the models successfully captured the variations in time-course activation dynamics, and the simulation results were fairly consistent with the experimental results for all three h cell derivatives. next, the egf ligand-dose response was examined to investigate how the l858r mutation affects cooperativity of egfr signaling. figure and additional file figure s1b show the experimental results in the phosphorylation levels of egfr, shc, mek, and erk in the egfr pathway when the dose of egf was varied. the phosphorylation levels of egfr and shc gradually increased as a function of the egf concentration in both h1299egfr-wt and h1299l858r cells, while mek and erk were highly phosphorylated even at egf concentrations as low as nm. the phosphorylation of egfr and shc in the h1299l858r cells showed a smaller dynamic range compared to that of the h1299egfr-wt cells, whereas there was no significant difference between the two cell types in mek and erk phosphorylation patterns. again, the l858r mutation reduced the phosphorylation levels in the upstream part of the signaling pathway in an egf-dose dependent manner, but had little effect on the downstream part. the simulation results imply that the models fairly reproduced the ligand-dose dependent behavior of the h1299egfr-wt and h1299l858r cells. also, both l858r models a and b seem to equally reproduce the time-course and the egf dose-dependency obtained with the h1299l858r cells. additional analyses are required to distinguish the model that represents the behaviors of the signaling dynamics associated with l858r mutation. mig plays a role in transmitting the effect of gefitinib to the downstream part of the egfr signaling pathway to clarify unknown other factors that affect gefitinib sensitivity, we investigated the gefitinib-dose response effect on the egfr signaling pathway. figure 4a and additional file figure s1c show the experimental results of phosphorylation of the signaling proteins in the presence of different doses of gefitinib. the h1299egfr-wt and h1299l858r cells showed similar dose-dependent responses in the upstream proteins egfr and shc. however, the h1299l858r cells were more sensitive to gefitinib administration at the phosphorylation of the downstream proteins, mek and especially erk, compared to the h1299egfr-wt cells, which were essentially insensitive to gefitinib for erk phosphorylation. figure 4b shows the simulation results of the gefitinib dose-response. the effect of the gefitinib was mimiced by changing the kinetic parameter k of egfr phosphorylation. α indicates the multiplying coefficient for k the results indicate that the l858r model a, including mig successfully reproduced the inhibitory effect for erk at higher concentrations of gefitinib, whereas model b, which included the effect of cbl, failed to reproduce this response. we confirmed that l858r model a with other parameter sets that have similar cost function values yields the same trends with regard to the role of mig in gefitinib sensitivity. also, we found that egfr-wt model at lower concentrations of egf is as sensitive to gefitinib for erk phosphorylation as l858r model a. based on these results, model b is inappropriate as the mechanism to explain the gefitinib sensitivity, whereas model a remains as a viable candidate. although the sensitivity to inhibition of phosphorylation by gefitinib was different in the downstream proteins between h1299egfr-wt and h1299l858r cells, the difference was small in the upstream proteins. therefore, it seemed likely that particular reaction steps in the pathway would amplify the small difference observed in the upstream proteins. to identify the critical steps, we analyzed the contribution of each parameter to the downstream phosphorylation influenced by gefitinib. we used an index that indicates the ratios of the upstream signaling activity to the downstream signaling activity in the presence of gefitinib. the output of the upstream signaling was the total phosphorylated shc, and the downstream output was the total phosphorylated erk. when rx is defined as the ratios of total phosphorylated x with gefitinib to total phosphorylated x without gefitinib, the index rshc/rerk was calculated for every combination of the four parameters affected by mig as a result, the combination of a lower k and k contributed to effectively inhibit the total phosphorylation of erk in the presence of gefitinib. the values in each panel indicate rshc/rerk calculated by using the values of parameters in l858r model a for two changing parameters and those in egfr-wt model for two unchanging parameters. the value of rshc/rerk for l858r model a is. next, we analyzed which downstream reactions were influenced by the combination of these two parameters. in this case, e11p/shcp/grb2/sos was used as the upstream output, and the downstream outputs were rasgtp, raf1a, mekp, mekpp, erkp, and erkpp. when ry is defined as the ratios of y with gefitinib to y without gefitinib, figure 5b shows re11p/shcp/grb2/sos/ry by varying the values of k and k. the values in each panel indicate re11p/shcp/grb2/sos/ry calculated by using the values of k and k in l858r model a and k and k in egfr-wt model. based on this analysis, we found high gefitinib sensitivity in mekpp, erkp, and erkpp. these results indicate that the steps of mek phosphorylation/dephosphorylation and erk phosphorylation/dephosphorylation amplify the gefitinib sensitivity. the combination of mig and gefitinib has a synergistic effect in inhibiting egfr signaling the only difference between the egfr-wt model and the l858r model a is in the negative egfr regulation produced by mig therefore, which can be experimentally verified by overexpressing mig we have known that expression level of mig is reversely correlated with erk phosphorylation level in the h derivatives with various egfr mutations. to study its effect on the upstream signaling, we performed western blotting for phosphorylated egfr. figure 6a shows that mig overexpression more inhibited egfr phosphorylation in the presence of gefitinib. therefore the effect of mig for gefitinib administration was further studied using mtt cell proliferation assay. at a high concentration of gefitinib, cell growth was suppressed in the cells with mig overexpression, but not in the h1299egfr-wt cells. this result indicates that mig indeed enhances the inhibitory effect of gefitinib as our mathematical model had predicted. we next analyzed the effects of a combinatorial perturbation of mig and gefitinib on the signaling inhibition. combinations of perturbations can be categorized into three interaction types: additive, antagonistic, or synergistic, according to whether the combination of two perturbations produces an effect equal to, less than, or larger than that expected based on the individual effects of the single perturbations. we analyzed the effect of the combination of mig and gefitinib on erkpp and found that the combination of mig and gefitinib exhibits a synergistic effect. this result indicates that synergism is produced by the dual inhibition of the step of egfr phosphorylation which both mig and gefitinib inhibit and the step of binding of egfr to shc which mig alone inhibits. discussion overexpression or mutation of egfr has been observed in lung cancers, and these molecular changes affect the prognosis and treatment sensitivity of patients. those abnormalities could cause changes in overall titers of signaling networks at the molecular, the cellular, and even the individual levels. mathematical model is helpful for understanding of the mechanical aspects of interconnected signaling network and predicting input-output behaviors in the pathway. however, it is often very difficult to explain the variation of reactants in signal transduction pathway using a single unified mathematical model. in this paper, we attempted to build such a unified model that could uncover the cell-specific regulatory mechanisms produced by overexpression and mutation of the egfr and the association with gefitinib sensitivity. the model used in this paper successfully reproduced the experimental observations concerning the activation of the key proteins in the pathway and discriminated the roles of mig and cbl in gefitinib sensitivity. the model was based on kinetic equations, and most of the parameters for these equations were common for all models. the differences in the parameters were confined to specific steps and proteins - egfr overexpression, inhibitory effects caused by mig and cbl overexpression leading to the degradation of egfr. our results revealed that the effectiveness of gefitinib in cells is largely affected by not only on its direct binding affinity with egfr but also on the presence of an additional molecule, mig according to recent reports, the sensitivity to kinase inhibition reflects intrinsic differences in the binding affinity of the egfr mutants such as l858r, g719s, and exon deletions. yun et al showed that gefitinib directly binds more tightly to the l858r mutant than to the wild type egfr in vitro, while fabian et al indicated that egfr with gefitinib sensitive mutations does not differ from wild type egfr in terms of gefitinib binding affinity. this would suggest that the stronger interaction of the mutated egfrs with gefitinib may not be the only one mechanism for the good clinical response to gefitinib in nsclc. this inconsistency in previous reports suggested to us that unknown factors may play an important role in gefitinib sensitivity. the gefitinib-dose response study showed that the difference in the phosphorylation levels of the downstream proteins inhibited by gefitinib is large between h1299egfr-wt and h1299l858r cells, whereas the difference in the upstream proteins is small. by a detailed computational analysis, we found that mig has an important role in propagating the gefitinib effect at the steps of mek phosphorylation/dephosphorylation and erk phosphorylation/dephosphorylation. additionally, the combination of the inhibition of egfr phosphorylation and the inhibition of binding of egfr to shc produced a synergistic inhibitory effect on egfr signaling. therefore, mig could be one of the critical factors to explain gefitinib sensitivity at cellular level. we constructed the model by referring to the earlier studies on mig functions. however, the model could be modified and improved when novel mechanism of mig in the regulation of egfr or new regulators associated with the egfr l858r mutation are identified by further studies. our results shown in figures and are consistent with a previously published report that egfr with the l858r mutation did not have stronger egfr phosphorylation upon egf stimulation when compared to the wild type egfr in h cells. on the other hand, guha et al and yun et al observed significantly high phosphorylation of the l858r-mutated egfr compared with wild type egfr expressed in hbec cells and sf cells. this inconsistency among cell types may be explained by the relative levels of mig which is highly expressed when egfr kinase is in an active state.  overall, the analysis presented in this paper allows understanding of the impacts of cancer-related abnormalities on the egfr signaling pathway. also, we demonstrate the feasibility of using computational models to predict one of the determinants for the evaluation of drug sensitivities. despite the fact that a new drug may help prevent the deaths of thousands of patients, there are many instances where the patients become severely ill or die because of serious unwanted side-effects. hence, in prescribing medications appropriate for individual patients, there is a clear need for guidance in predicting side-effects and drug sensitivity. it would be no exaggeration to say that the side-effects could not be predicted in advance, since signaling pathways are very complex. we believe that in part such guidance can be predicted by computational modeling of appropriate signaling pathways. authors' contributions yn carried out the mathematical modeling and analysis work, and wrote the manuscript. tnag helped to draft the manuscript. ru, ys, and sft carried out the experimental laboratory work. tnakak and tnaka proposed a basic idea of the mathematical methods. ht participated in the design and coordination of the study. all of the work was supervised by mo. all authors have read and approved the final version of the manuscript. supplementary material additional file 1 supplementary information. this pdf file contains all additional figures and tables referenced in the text. click here for file additional file 2 h model. this xml file contains our model represented by sbml. click here for file acknowledgements we are grateful to dr yi-robg chen for providing h derivative cell lines for using in the present paper. we also thank prof tadashi masuda, dr kaoru mogushi, and dr takeshi hase for a critical reading of the manuscript and helpful discussions. all parallel computation was performed by using the riken integrated cluster of clusters facility.
epidermal growth factor receptor signaling plays an important role in the regulation of cell proliferation, survival, metastasis, and invasion in various tumors. we predicted and experimentally verified that mig which is a known negative regulator of egfr and specifically expressed in h1299l858r cells, synergized with gefitinib to suppress cellular growth. thus, we showed that l858r receptor mutation in combination with expression of its negative regulator, mig alters signaling outcomes and results in variable drug sensitivity. in this paper, we performed experimental and computational analyses to clarify the underlying mechanism of egfr signaling and cell-specific gefitinib responsiveness in three h1299-derived nsclc cell lines; h wild type, h with an overexpressed wild type egfr, and h with an overexpressed mutant egfr l858r. computational analyses indicated that this inhibitory effect is amplified at the phosphorylation/dephosphorylation steps of mek and erk. earlier studies showed that the egfr is frequently overexpressed in non-small-cell lung cancer and egfr mutations at specific amino acid residues in the kinase domain induce altered responsiveness to gefitinib, a small molecule egfr tyrosine kinase inhibitor.
 epidermal growth factor receptor signaling plays an important role in the regulation of cell proliferation, survival, metastasis, and invasion in various tumors. earlier studies showed that the egfr is frequently overexpressed in non-small-cell lung cancer and egfr mutations at specific amino acid residues in the kinase domain induce altered responsiveness to gefitinib, a small molecule egfr tyrosine kinase inhibitor. however, the mechanism underlying the drug response modulated by egfr mutation is still largely unknown. to elucidate drug response in egfr signal transduction pathway in which complex dynamics of multiple molecules involved, a systematic approach is necessary. in this paper, we performed experimental and computational analyses to clarify the underlying mechanism of egfr signaling and cell-specific gefitinib responsiveness in three h1299-derived nsclc cell lines; h wild type, h with an overexpressed wild type egfr, and h with an overexpressed mutant egfr l858r.  we predicted and experimentally verified that mig which is a known negative regulator of egfr and specifically expressed in h1299l858r cells, synergized with gefitinib to suppress cellular growth. computational analyses indicated that this inhibitory effect is amplified at the phosphorylation/dephosphorylation steps of mek and erk.  thus, we showed that l858r receptor mutation in combination with expression of its negative regulator, mig alters signaling outcomes and results in variable drug sensitivity. 