MEK Inhibition Leads to PI3K/AKT Activation by Relieving a ...Molecular and Cellular Pathobiology...

Molecular and Cellular Pathobiology

MEK Inhibition Leads to PI3K/AKT Activation by Relievinga Negative Feedback on ERBB Receptors

Alexa B. Turke1,2, Youngchul Song1, Carlotta Costa1, Rebecca Cook4, Carlos L. Arteaga4, John M. Asara2,3,and Jeffrey A. Engelman1,2

AbstractThe phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated in a wide

range of human cancers. In many cases, concomitant inhibition of both pathways is necessary to blockproliferation and induce cell death and tumor shrinkage. Several feedback systems have been described inwhich inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, therebydecreasing the effectiveness of single-agent targeted therapies. In this study, we describe a feedback mechanismin which MEK inhibition leads to activation of PI3K/AKT signaling in EGFR and HER2-driven cancers. We foundthatMEK inhibitor–induced activation of PI3K/AKT resulted fromhyperactivation of ERBB3 as a result of the lossof an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2.Mutation of this amino acid led to increased ERBB receptor activation and upregulation of the ERBB3/PI3K/AKTsignaling pathway, which was no longer responsive to MEK inhibition. Taken together, these results elucidatean important, dominant feedback network regulating central oncogenic pathways in human cancer. CancerRes; 72(13); 3228–37. �2012 AACR.

IntroductionThe phosphoinositide 3-kinase (PI3K), RAF/MEK/ERK

mitogen-activated protein kinase (MAPK), and mTORC1pathways transmit signals from receptor tyrosine kinases(RTKs) to downstream effector networks regulating cellgrowth, metabolism, survival, and proliferation (1–3).Numerous feedback systems regulating these oncogenicpathways have been described, and can potentially impactthe sensitivity of cancers to kinase inhibitors. For example,inhibition of mTORC1 relieves proteasomal degradation ofIRS-1 leading to feedback up-regulation of IRS-1/PI3K/AKT,reducing the efficacy of mTORC1 inhibitors as single agentsand prompting the use of combination therapies (4–6). PI3Kand AKT inhibitors relieve a negative feedback on ERBBreceptors and other RTKs leading to partial reactivation ofPI3K/AKT signaling, MEK/ERK signaling, and other down-stream pathways, potentially limiting the use of PI3K inhi-bitors as single agents (7–9).

Targeted therapies, such as the EGFR inhibitors gefitiniband erlotinib, are highly effective when cells are "addicted," andinhibition of the target leads to downregulation of criticalgrowth and survival signaling pathways, especially PI3K/AKTandMEK/ERK (10–12). We recently found that treatment witha combination of a MEK inhibitor and a PI3K inhibitor led tosignificant apoptosis in EGFR-driven cancers, similar to thatinduced by an EGFR TKI, whereas treatment with eitherpathway inhibitor alone did not induce marked cell death(11). In those studies, treatment with a single-agent MEKinhibitor led to increased AKT phosphorylation. Indeed, sev-eral other studies have shown that MEK inhibition leads toincreased AKT activation, often resulting in reduced efficacy ofMEK inhibitors as single agents (11, 13–15). However, themolecular mechanisms underlying this feedback remainunknown.

Several mechanisms for MEK feedback regulation of AKTsignaling have been suggested. For example, ERK-mediatedserine phosphorylation of theGAB1 adaptor has been shown tonegatively regulate GAB1–PI3K binding and downstream AKTsignaling (16–18). MEK inhibition can also downregulatemTORC1 signaling, relieving negative feedback on IGF-IR/IRS-1 and activating PI3K/AKT signaling (19). ERK has alsobeen shown to directly regulate ERBB tyrosine phosphoryla-tion (20, 21). However, it remains unclearwhichmechanisms, ifany, are dominant inMEK inhibitor–induced activation of AKTsignaling in EGFR or HER2-driven cancers. As multiple MEKand BRAF inhibitors, including the highly selective allostericMEK1/2 inhibitor, AZD6244 (22), are being developed, under-standing the signaling feedbacks induced by MEK inhibitorsthat may ultimately impact their use will become increasinglyimportant.

Authors' Affiliations: 1Massachusetts General Hospital Cancer Center;2Department of Medicine, Harvard Medical School; 3Division of SignalTransduction, Beth Israel Deaconess Medical Center, Boston, Massachu-setts; and 4Vanderbilt-Ingram Cancer Center, Nashville, Tennessee

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Jeffrey A. Engelman, Massachusetts GeneralHospital Cancer Center, Building 149, 13th St, Boston, MA 02129. Phone:617-724-7298; Fax: 617-724-9648; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3747

�2012 American Association for Cancer Research.

CancerResearch

Cancer Res; 72(13) July 1, 20123228

on April 5, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 2, 2012; DOI: 10.1158/0008-5472.CAN-11-3747

http://cancerres.aacrjournals.org/

In this study, we examined the molecular mechanism bywhich MEK inhibition leads to increased AKT phosphorylationin EGFR and HER2-driven cancers. We provide evidence sug-gesting that this feedback occurs at the level of increasedphosphatidylinositol 3,4,5-trisphosphate (PIP3) induced by anincreased association between ERBB3 and PI3K. IncreasedERBB3 activation results from loss of an inhibitory ERK-depen-dent threonine phosphorylation in the conserved juxtamem-brane domains of EGFR andHER2, previously found to regulateto EGFR auto-phosphorylation (21). Elucidation of this mech-anismprovidesa greaterunderstandingof the feedback systemsregulating key pathways that drive human cancers.

Materials and MethodsCell culture reagents and Western blot analysesCell lines, inhibitors, and growth conditions are described in

Supplementary Materials and Methods. Cells were lysed in anNP-40 containing buffer, separated by SDS/PAGE, and trans-ferred to PVDF membranes. Antibody binding was detectedusing enhanced chemiluminescence (Perkin-Elmer).

Biotin labeling and immunoprecipitationHCC827 cells werewashedwith PBS and labeled for 1 hour at

4�C in 0.5 mg/mL Sulfo-NHS-LC-Biotin (Thermo Scientific)resuspended in PBS � AZD6244. Labeling was quenched with100mmol/L glycine. Cells were then returned tomedia at 37�Cbefore lysis. Biotin-labeled cell surface proteins were immu-noprecipitated with NeutrAvidin Agarose Resins (ThermoScientific), separated by SDS-PAGE, and immunoblotted todetect the indicated proteins. Transferrin receptor was used asa loading control.

Xenograft studiesXenograft studies were conducted in accordance with the

standards of the Institutional Animal Care and Use Committee(IACUC) under a protocol approved by the Animal Care andUse Committee ofMassachusetts General Hospital. Nudemice(nu/nu; 6–8 weeks old; Charles River Laboratories) wereinjected with a suspension of 5 � 106 H1975 cells subcutane-ously into the flank of each mouse. Once the mean tumorvolume reached�500mm3, AZD6244was administered by oralgavage in 3 doses of 25 mg/kg over 30 hours.

PIP2/PIP3 quantificationPhospholipids were isolated from cells and PIP3 and PI(4,5)

P2 levels were measured using ELISA kits (Echelon, K-2500sand K4500) according to the manufacturer's instructions.Statistical significance was calculated using a t test.

Quantitative reverse transcriptase-polymerasechain reactionRNA was isolated using the RNEasy Kit (Qiagen) and cDNA

was transcribed with Superscript II Reverse Transcriptase(Invitrogen) and used as a template for PCR amplifications.The relative copy number for ERBB3 and HRGwas determinedusing quantitative reverse transcriptase-polymerase chainreaction (qRT-PCR) using a light-cycler 480 (Roche) as previ-

ously described (11). The PCR primers and conditions areavailable upon request.

siRNA and transient transfectionsHCC827 and BT-474 cells were transfected with 50 nmol/L

ERBB3s4779 silencer select validated siRNA or negative control(Ambion) with HiPerFect Transfection Reagent (Qiagen)according to manufacturer's instructions.

Transient transfections of CHO-KI cells were conductedwith TransIT-LT1 Transfection Reagent (MirusBio LLC)according to the manufacturer's recommendations. Wild-typeERBB3 was cotransfected with an equal ratio of GFP or wild-type or mutant EGFR or HER2.

shRNA, DNA constructs, and lentiviral productionHCC827 cells were infected with shERBB3 as previously

described (23, 24) with tet-on PLKO shERBB3 or scrambleshRNA knockdown vectors (Addgene) and selected in puro-mycin. shRNA hairpin sequences are provided in the Supple-mentary Materials and Methods.

Human EGFR (wild-type and exon 19del) and HER2 cDNAcoding regions were cloned into the pENTR/D-TOPO vector(Invitrogen) andmutants were constructedwithQuick ChangeSite-Directed Mutagenesis Kit (Stratagene) according to themanufacturer's instructions. All constructs were confirmed byDNA sequencing. Constructs were cloned into the plenti-IRES-GFP lentiviral vector (Addgene) and infectionswere conductedas described previously (25).

Flow cytometryBT-474 cells were transfected with ERBB3 siRNA (Ambion)

for 48 hours, then treated with AZD6244 or GDC-0941 for72 hours. Cells were collected and stained with propidiumiodide (PI) and Annexin V as described previously (26). Cellswere analyzed using a BD LSR3 analytical flow cytometer(BD Biosciences). Apoptosis was calculated using the sumof Annexin V–positive and PI/Annexin V double-positivecells.

Tandem mass spectrometryEGFR or HER2 was immunoprecipitated from cells treated

with AZD6244 using anti-EGFR antibody (Santa Cruz) or ananti-HER2 antibody, separated by SDS/PAGE, stained withCoomassie blue. Bands were excised and samples were pre-pared and analyzed by reversed-phase microcapillary/tandemmass spectrometry (LC/MS-MS) as described previously(27–29) and further detailed in the Supplementary Materialsand Methods.

ResultsMEK inhibition leads to activation of ERBB3/PI3K/AKT

We previously observed that AKT phosphorylationincreased in response to MEK inhibition in HER2-amplifiedand EGFR-mutant cancer cells (11). To determine whetherthis potential feedback is observed in multiple EGFR- orHER2-addicted cancer models, we treated HER2-amplified orEGFR-mutant cell lines with the highly selective allosteric

MEK Inhibitor–Induced Feedback on ERBB Receptors

www.aacrjournals.org Cancer Res; 72(13) July 1, 2012 3229




MEK1/2 inhibitor, AZD6244. This MEK inhibitor was used ata concentration of 2 mmol/L, which sufficiently inhibitedERK1/2 phosphorylation in the HCC827 cell line (Supple-mentary Fig. S1). Similar results were observed using 2distinct allosteric MEK inhibitors, GSK212 and PD0325901(Supplementary Fig. S1). In each cell line, we observedincreased AKT phosphorylation at both S473 and T308 afterAZD6244 treatment, as well as increased phosphorylation ofseveral AKT targets including GSK3a/b, ATP citrate lyase,and PRAS40 (Fig. 1A). We confirmed that these proteinswere AKT substrates, as cotreatment with an allosteric AKTinhibitor blocked their phosphorylation (Supplementary Fig.S2). MEK inhibition also led to upregulation of phospho-CRAF and phospho-MEK (Fig. 1A), suggesting activation of acommon upstream signaling molecule. This feedback alsooccurred in vivo, as we observed increased phospho-AKT inan EGFR-mutant H1975 (L858R/T790M) xenograft modeltreated with AZD6244 (Fig. 1B).

Increased AKT phosphorylation suggested a potentialincrease in the abundance of PIP3 (the lipid product of PI3K).

Therefore, EGFR-driven HCC827 and HER2-driven MDA-MB-453 cells were treated with a MEK inhibitor, lipids wereisolated, and PIP3 levels were quantified. In both cell lines,AZD6244 induced significant increases in PIP3 (Fig. 1C).Wedidnot observe any change in expression of the PTENphosphataseresponsible for de-phosphorylating PIP3 after MEK inhibition(Fig. 1A). To determine if MEK inhibition led to activation ofPI3K, we immunoprecipitated the p85 regulatory subunit ofPI3K and assessed the abundance of bound adaptors. PI3Kconsists of a p110 catalytic subunit and a p85 regulatorysubunit, and is activated when p85 SH2 domains bind totyrosine-phosphorylated proteins with YXXM motifs. Treat-ment with AZD6244 increased the association between PI3Kand tyrosine-phosphorylated adaptors, including ERBB3 andGAB1 (Fig. 1D). These results suggest thatMEK inhibition leadsto an increase in the phospho-tyrosine signaling cascades thatdirectly activate PI3K.

In EGFR- and HER2-driven cancers, ERBB3 is a primaryactivator of PI3K/AKT (24, 30). We observed increasedERBB3 binding to PI3K after MEK inhibition (Fig. 1D), and

p-AKT (473)

p-AKT (308)

AKT

p-ERK

ERK

p-MEK (217/221)

p-CRAF (338)

p-GSK3α

p-GSK3β

p-PRAS40 (246)

p-ATPCitrateLyase (455)

AKTsubstrates

- + - + - + - + - + - + AZD6244

A

C D

p-AKT (308)

p-AKT (473)

AKT

p-ERK

ERK

Actin

PTEN

p-ERBB3 (1289)

ERBB3

p-ERBB3 (1289)

ERBB3

NRDP1

Actin

B

150

250

100

p-Tyr

ERBB3

IP: p85IgG

- - + AZD6244

IRS-1

GAB1

p85

ERBB3IRS-1

GAB1

375

350

325

300

275

250

225

200

175

150

125

100

75

50

25

0

MDA-MB-453 HCC827

Control 6 h AZD 6 h AZD18 h AZD Control 18 h AZD

PIP

3/P

I(4

,5)P

2 (

%)

Figure 1. MEK inhibition leads tofeedback activation of ERBB3/PI3K/AKT signaling. A, EGFR-addicted (HCC827, H4006, H1975)or HER2-addicted (BT-474, NCI-N87, MDA-MB-453) cell lines weretreated with 2 mmol/L AZD6244 for6 hours. Cell lysates wereimmunoblotted to detect theindicated proteins. B, H1975(EGFR L858R/T790M) xenograftswere treated with AZD6244 25 mg/kg twice a day. Mice weresacrificed 6 hours after treatmentand tumors were harvested. Tumorcell lysates were immunoblotted todetect the indicated proteins. C,MDA-MB-453 and HCC827 cellswere treated for 6 or 18 hourswith 2mmol/L AZD6244. Phospholipidswere isolated from cell lysates andrelative PIP3 and PI(4,5)P2 levelswere quantified by ELISA. Eachdata point represents the mean �SEMof 2 independent experimentscarried out in triplicate. Fornormalization, the PIP3/PI(4,5)P2

ratio was set to 100% for untreatedcells. �, P < 0.05. D, HCC827 cellswere treated for 6 hours with2 mmol/L AZD6244. Cell extractswere immunoprecipitated (IP) withan anti-p85 antibody followed byWestern blot analysis with anti-p-Tyr and anti-p85 antibodies. Blotswere stripped and reprobed withantibodies specific to ERBB3,GAB1, and IRS-1.

Turke et al.

Cancer Res; 72(13) July 1, 2012 Cancer Research3230




accordingly, MEK inhibition substantially increased tyro-sine-phosphorylated ERBB3 levels (Fig. 1A). In some celllines, we observed an increase in total ERBB3 along withphospho-ERBB3 (Fig. 1A). Of note, we did not observe achange in expression of the E3-ubiquitin ligase, neuregulinreceptor degradation protein 1 (NRDP1), which can controlthe steady-state levels of ERBB3 (refs. 31, 32; Fig. 1A). Therewas also no increase in ERBB3 mRNA levels after AZD6244treatment (Supplementary Fig. S3), suggesting that anyincrease in ERBB3 protein levels is posttranscriptional.To assess the kinetics of this feedback response, we

treated the HCC827 cells with AZD6244 over a time course.Phoshosphorylation of ERBB3 and AKT, as well as down-stream substrates, increased after just 1 hour of MEKinhibition and continued to accumulate for 24 hours(Fig. 2A). To determine if the feedback activation of ERBB3occurs on the plasma membrane, we biotin-labeled thesurface of HCC827 cells in the presence or absence ofAZD6244 and immunoprecipitated the labeled proteins.After just 1 hour of MEK inhibition during biotin labeling,surface levels of the activated receptor were substantiallyelevated (Fig. 2B). Total ERBB3 on the cell surface alsoincreased after AZD6244 treatment. MEK inhibition did notseem to significantly affect the kinetics of loss of ERBB3on the cell surface (Fig. 2B), suggesting that receptor inter-nalization or cycling was not significantly affected. Thesedata show that feedback activation of ERBB3 occurs rapidlyon the plasma membrane.

Knockdown of ERBB3 abrogates MEK/ERK feedback onAKT and downstream substrates

To determine if increased ERBB3 phosphorylation causedthe increase in AKT phosphorylation after MEK inhibition, wesuppressed expression of ERBB3 using a Tet-inducibleshERBB3 hairpin construct. After treatment with doxycyclinethere was effective knockdown of ERBB3, and this abrogatedthe increase in AKT signaling normally observed after MEKinhibition (Fig. 3A). In HER2-amplified BT-474 cells withabrogated ERBB3 expression, the increase in AKT signalingafter MEK inhibition was also attenuated (Fig. 3B). In contrastto HCC827 cells, we observed significant downregulation ofbasal AKT phosphorylation in BT-474 cells after ERBB3 knock-down (Fig. 3B), indicating the sole reliance on ERBB3 for PI3Kactivation in this HER2-amplified cancer. In contrast, EGFR-mutant cancers also use GAB1 to activate PI3K (Fig. 1D;refs. 24, 33).

We suspected that knockdown of ERBB3 may increase theefficacy of MEK inhibition by suppressing PI3K/AKT signaling.Treatment with ERBB3 siRNA induced similar levels of celldeath compared with treatment with a PI3K inhibitor, GDC-0941 (Fig. 3C). Indeed, combining ERBB3 siRNA with AZD6244increased the cell death response, approaching the level ofapoptosis achieved with GDC-0941 in combination withAZD6244 (Fig. 3C). These data indicate that ERBB3 plays asignificant role in MEK feedback on PI3K/AKT signaling inEGFR- andHER2-driven cell lines, suggesting that combinationtherapies targeting MEK and ERBB3 or MEK and PI3K may

Figure 2. Feedback activation andincreased surface localization of ERBB3occurs within 1 hour of treatment withAZD6244. A, HCC827 cells weretreated for the indicated number of hourswith AZD6244 (2 mmol/L). Cell lysates wereimmunoblotted to detect the indicatedproteins. B, HCC827 cells were treated withdimethyl sulfoxide (DMSO; NoRx) orAZD6244 (2 mmol/L) for 1 hour at 4�C duringbiotin labeling of surface proteins. Afterlabeling, cells were returned to media(�AZD6244) and treated for the indicatednumber of hours before lysis. Biotin-labeledsurface proteins were immunoprecipitated(IP) with NeutrAvidin beads, separated bySDS-PAGE, and immunoblotted to detectthe indicated proteins. WB,Western blot.

A

B

p-ERBB3

ERBB3

Transferrin receptor

IP: NeutrAvidin

WB:0 4 8 24 0 4 8 24 h after

biotin surface labeling

No Rx AZD6244

p-ERBB3 (1289)

ERBB3

0 1 6 16 24 h AZD6244

p-AKT (473)

p-AKT (308)

AKT

p-ERK

ERK

p-PRAS40 (246)

p-ATPCitrateLyase (455)

PHLPP

Actin

HCC827






block feedback activation of ERBB3/PI3K/AKT signaling andthus be more effective than treatment with a MEK inhibitoralone.

MEK inhibition results in feedback activation of ERBB3in KRAS-mutant cell lines with low basal levels ofphospho-ERBB3

We next determined whether MEK feedback on ERBB3 alsooccurs in cancers not addicted to EGFR or HER2. We treated apanel of KRAS-mutant cell lines, which have low basal levels ofphospho-ERBB3 with AZD6244. Surprisingly, MEK inhibitionled to significant activation of ERBB3, but in contrast to EGFR-mutant and HER2-amplified cancers, the increased ERBB3activation did not translate to increased phospho-AKT(Fig. 4A). Similar to the EGFR- and HER2-driven models, wealso observed upregulation of phospho-CRAF and phospho-MEK after MEK inhibition. We suspect that increased ERBB3phosphorylation did not drive PI3K in these KRAS-mutant celllines because they express significantly less EGFR and HER2,resulting in markedly lower levels of phospho-ERBB3 com-pared with those observed in EGFR- and HER2-driven models(Supplementary Fig. S4). Indeed, we recently reported thatIGF-IR/IRS signaling is the major PI3K input in these cells(19). Thus, the feedback from MEK inhibition to activationof ERBB3 seems to be conserved in all 3 of the modelswe tested, including EGFR-mutant, HER2-amplified, andKRAS-mutant cancers, but results in increased PI3K/AKT

signaling only in cells that express sufficient absolute levelsof phospho-ERBB3.

The feedback observed in EGFR- and HER2-driven cancersis distinct from a well-described feedback mechanism inwhich mTORC1 inhibition leads to increased IRS-1 expres-sion and upregulation of IGF-IR/IRS signaling (5, 6). In theKRAS-mutant cell lines that we analyzed, which primarilyuse IGF-1R/IRS to activate PI3K (19), treatment with themTORC1 inhibitor rapamycin led to feedback activation ofAKT signaling that was blocked by cotreatment with theIGF-IR/IR inhibitor, NVP-AEW541 (Fig. 4A and B). In con-trast, MEK inhibitor–induced activation of ERBB3 in theKRAS-mutant cancers was blocked by gefitinib, but not byNVP-AEW541 (Fig. 4B). Accordingly, NVP-AEW541 failed toabrogate AZD6244-induced activation of phospho-AKT inEGFR- and HER2-driven cell lines (Supplementary Fig. S5).Of note, we have also previously observed cancers in whichMEK inhibition leads to inhibition of downstream phospho-S6, resulting in feedback activation of IGF-IR/IRS-1/AKTsignaling independent of ERBB3 in both KRAS wild-type andmutant cancers (19), suggesting that cancers not driven byEGFR or HER2 may have alternate, ERBB3-independent,mechanisms of MEK inhibitor–induced feedback activationof AKT. Our data suggest that the effect of MEK inhibition onERBB3 is a novel feedback mechanism, distinct frommTORC1 feedback on IGF-IR/IRS-1. A model describingthese findings is shown in Fig. 4C.

HCC827 shERBB3-Tet–inducible cells

p-ERBB3

ERBB3

p-AKT (473)

p-AKT (308)

AKT

p-ERK

p-ATPCitrateLyase

Actin

Control +Dox

0 1 6 16 24 0 1 6 16 24 h AZD6244

Cont siERBB3

- + - + AZD6244 6 h

BT-474

A

B

p-ERBB3

ERBB3

p-AKT (473)

p-AKT (308)

AKT

p-ERK

ERK

p-GSK3α

p-GSK3β

p-ATPCitrateLyase

C 35

30

25

20

15

10

5

0

Control

AZD6244

siERBB3

siERBB3 +

AZD6244

GDC-0

941

GDC-0

941 + A

ZD6244

% A

popto

sis

Figure 3. Suppression of ERBB3expression abrogates MEK/ERKfeedback on PI3K/AKT signaling.A, HCC827 cells infected with alentivirus expressing a tetracycline(Tet)-inducible shERBB3 hairpinwere induced with 100 ng/mLdoxycycline (þDox) for 48 hours.After knockdown, cells weretreated with AZD6244 (2 mmol/L)for the indicated number of hours.Cell lysateswere immunoblotted todetect the indicated proteins. B,BT-474 cells were transfected withcontrol or ERBB3-targeted siRNAfor 48 hours, followed by treatmentwith AZD6244 for 6 hours. Celllysates were immunoblotted withthe indicated proteins. C, BT-474cells were transfected with controlor ERBB3-targeted siRNA for 48hours, followed by treatment with 2mmol/L AZD6244, 1 mmol/L of thePI3K inhibitor GDC-0941, or thecombination for 72 hours. Cellswere collected and stained forpropidium iodide and Annexin V todetermine the percentage ofapoptotic cells. Each data pointrepresents the mean � SD of 3independent experiments.

Turke et al.





MEK inhibition results in increased tyrosinephosphorylation of ERBB3 due to inhibition of ERK-mediated threonine phosphorylation of EGFR and HER2We investigated themechanism leading to increased ERBB3

phosphorylation after MEK inhibition. HRG ligand expressionwas not increased with AZD6244 (Supplementary Fig. S6);however, MEK inhibitor–induced feedback activation of AKTrequired EGFR or HER2 kinase activity (SupplementaryFig. S7). Indeed, even in KRAS-mutant SW1463 cells, MEKfeedback on ERBB3 was still dependent on EGFR kinaseactivity (Fig. 4B).Because EGFR and HER2 inhibition blocked MEK feedback

activation of ERBB3/PI3K/AKT, we investigated whether MEKinhibition affected the activation of these receptors. Treatmentof HCC827 and BT-474 cells with AZD6244 resulted in

increased tyrosine phosphorylation of both EGFR and HER2,indicative of receptor activation (Fig. 5A). It has been reportedthat activation of EGFR involves the formation of an asym-metric dimer (34). Formation of the active RTK dimer isfacilitated by stabilizing contacts made between the juxta-membrane domain of the "receiver/acceptor" kinase and theC-terminal lobe of the "activator/donor" kinase (21, 34, 35).Threonine 669 of EGFR, a putative MAPK target site, isconserved within the juxtamembrane domains of EGFR, HER2,and ERBB4 (21). When overexpressed in CHO-KI cells, muta-tion of this threonine site has been shown to augment EGFRtyrosine phosphorylation (20, 21, 36, 37). However, the phys-iological consequences of EGFR T669 phosphorylation incancer models and on ERBB3/PI3K/AKT signaling remainedunknown.

Figure 4. MEK/ERK feedback onERBB3 occurs in KRAS-mutant celllines and is distinct from TORC1feedback on IRS-1. A, KRAS-mutantcell lines were treated with AZD6244(2 mmol/L) or rapamycin (50 nmol/L)for 6 or 24 hours. Cell lysates wereimmunoblotted to detect theindicated proteins. B, SW1463 cellswere treated with 1 mmol/L of theEGFR inhibitor gefitinib or 1mmol/Lofthe IGF-IR inhibitor NVP-AEW541,alone or in combinationwith 2mmol/LAZD6244 or 50 nmol/L rapamycinfor 24 hours. Cell lysates wereimmunoblotted to detect theindicated proteins. C, model of MEKfeedback on ERBB3 in EGFR/HER2-addicted cancers (top) and KRAS-mutant cancers with low phospho-ERBB3 (bottom).

p-ERK

ERK

p-S6 (240/244)

S6

p-AKT (473)

p-AKT (308)

AKT

p-MEK

p-C-RAF

p-ERBB3

ERBB3

SW1463 Gp5d SW837 BA

C

p-ERBB3

ERBB3

p-AKT (308)

p-AKT (473)

AKT

p-ERK

ERK

p-MEK

Actin

SW1463

RapamycinAZD6244NVP-AEW541

Gefitinib- +- - - -+- - - - + - +

+- - + + - -+- - +- - +

ERBB3 IGFRIRS-1

AKT

MEK

TORC1ERK

AZD6244

EGFR/HER2-driven

ERBB3

MEK

ERK

AZD6244

AKT

TORC1

IGFRIRS-1

KRAS-mutant (low basal phospho-ERBB3)

ERBB3

AKT

MEK

TORC1ERK Rapamycin

IGFRIRS-1






We hypothesized that the MEK/ERK pathway may suppresstrans-phosphorylation of ERBB3 by directly phosphorylatingthe juxtamembrane domains of EGFR and HER2, and that thiscould be a dominant MEK inhibitor–induced feedback leadingto AKT activation in these cancers. We used tandem massspectrometry to measure the effects of AZD6244 on phosphor-ylation of this juxtamembrane domain threonine residue inboth EGFR-mutant and HER2-amplified cancer models. Tar-geting both the phosphorylated and nonphosphorylated pep-tide forms, we detected a 66% average decrease in EGFR T669phosphorylation and a 75% decrease in HER2 T677 phosphor-ylation upon treatment with AZD6244 (Fig. 5B; SupplementaryFig. S8). Phospho-specific antibodies confirmed that treatmentwith AZD6244 inhibited phosphorylation of T669 of EGFR andthe analogous T677 of HER2 (Fig. 5A). Together these dataindicate that loss of this inhibitory threonine phosphorylationon the juxtamembrane domains of EGFR and HER2 occurs incancer cell lines after MEK inhibition.

Mutation of T669 and T677 abrogates MEK-inducedsuppression of ERBB3 activation

We hypothesized that MEK inhibition activates AKT byinhibiting ERK activity, which blocks an inhibitory threoninephosphorylation on the juxtamembrane domains of EGFR and

HER2, thereby increasing ERBB3 phosphorylation. To test thishypothesis, we transiently transfected CHO-KI cells, which donot express ERBB receptors endogenously, with wild-typeERBB3 with either wild-type EGFR or EGFR T669A. In cellstransfected with wild-type EGFR, MEK inhibition led to feed-back activation of phospho-ERBB3 and phosho-EGFR, reca-pitulating the results we had observed in our panel of cancercell lines (Fig. 6A). In contrast, the EGFR T669A mutantincreased both basal EGFR and ERBB3 tyrosine phosphor-ylation that was not augmented by MEK inhibition. As acontrol, we treated CHO-KI cells expressing EGFR T669Awith HRG ligand to induce maximal ERBB3 phosphorylation(Fig. 6A), indicating that the lack of induction of phospho-ERBB3 in EGFR T669A expressing cells after MEK inhibitionwas not simply because of the saturation of the system withphospho-ERBB3. We observed analogous results in CHO-KIcells expressing wild-type ERBB3 in combination with wild-type or T677A mutant HER2 (Fig. 6B). Together these resultssupport the hypothesis that inhibition of ERK-mediatedphosphorylation of a conserved juxtamembrane domainthreonine residue leads to feedback activation of EGFR,HER2, and ERBB3 (Fig. 7).

To determine if this feedback model explains the activationof PI3K signaling in EGFR-mutant cancers, we used shRNA toknockdown endogenous EGFR (which carries an exon 19deletion) in the HCC827 NSCLC cell line and replaced witheither EGFR (exon 19del) wild-type at T669, or EGFR (exon19del) carrying a T669A mutation. Of note, this is the sameEGFR-mutant cell line in which we observed that EGFR T669 isphosphorylated in MEK-dependent manner (Fig. 5; Supple-mentary Fig. S8A). When endogenous EGFR was replaced withEGFR (exon19del) wild-type at T669, MEK inhibition led tosignificant feedback activation of ERBB3/PI3K/AKT signaling(Fig. 6C). However, replacement with the EGFR (exon19 del)T669A mutant led to increased tyrosine phosphorylation ofboth EGFR and ERBB3, and activation of PI3K/AKT signaling,mimicking the effect of MEK inhibition (Fig. 6C). As expected,addition of AZD6244 failed to further augment ERBB3 andAKTphosphorylation in cells expressing the 669A mutant. Theseresults show that EGFR T669 phosphorylation is necessary forMEK/ERK to suppress EGFR-mediated activation of ERBB3.This supports the hypothesis that a dominant ERK feedback onERBB3/PI3K/AKT is mediated through phosphorylation ofT669 on EGFR (or T677 HER2).

DiscussionRAF and MEK inhibitors are being developed as treatments

for cancers with activation of RAF/MEK/ERK signaling. How-ever, with the exception of BRAF-mutant melanomas, theefficacy of these drugs as single agents has been underwhelm-ing to date. Although there are several potential reasons for thislack of efficacy, feedback activation of parallel oncogenicpathways including PI3K/AKT has been invoked (11, 13–15).This idea is analogous to findings that mTORC1 inhibitors arelimited by feedback activation of PI3K signaling (4, 6). In thisstudy, we observe that MEK-inhibitor–induced activationof PI3K/AKT occurs in multiple ERBB-driven cancer modelsvia loss of an inhibitory threonine phosphorylation in the

p-EGFR (1068)

p-EGFR (669)

EGFR

p-ERK

ERK

Actin

p-HER2 (1221/2)

p-HER2 (677)

HER2

p-ERK

ERK

Actin

HCC827 BT-474

A

B 1.5

1.0

0.5

0.0

EGFR T669

HCC827 cells

HER2 T677

BT-474 cells

Con

trol

AZD62

44

Con

trol

AZD62

44

No

rma

lize

d r

atio

ph

osp

ho

-pe

ptid

e s

ign

al

Figure 5. MEK inhibition blocks phosphorylation of a direct ERK target sitein the conserved juxtamembrane domains of EGFR and HER2. A,HCC827 and BT-474 cells were treated with AZD6244 (2 mmol/L) for 6 or24 hours. Cell lysates were immunoblotted to detect the indicatedproteins. Phospho-HER2 (677) runs at 185 kDa and is recognized by theanti-phospho-EGFR (669) antibody. B, HCC827 or BT-474 cells weretreated with AZD6244 (2 mmol/L) for 6 hours. EGFR or HER2 wasimmunoprecipitated, and Coomassie blue bands were excised forquantitative mass spectrometric analysis of peptide phosphorylation.Eachdatapoint represents themean�SDof 3 independent experiments.

Turke et al.





conserved juxtamembrane domains of EGFR and HER2. Phos-phorylation of this threonine residue has been shown to impairEGFR activation, likely through disruption of receptor dimer-ization (21).Our findings suggest that direct ERK-mediated phosphor-

ylation of EGFR T669 and HER2 T677 suppresses activation of

ERBB3. These findings agree with those by Li and colleagueswho observed that MEK inhibition failed to increase phos-phorylation of EGFR T669A homodimers expressed in CHO-KI cells (20). In this study, we extend previous findingsby directly showing the effects of EGFR T669A on ERBB3/PI3K/AKT signaling in an EGFR-mutant cancer cell line.

Figure 6. T669A mutation of EGFRor T677A mutation of HER2 blocksMEK feedback activation of ERBBreceptors. A and B, CHO-KI cellswere transiently transfected toexpress wild-type ERBB3 incombination with a GFP control, orwild-type or mutant EGFR (A) orHER2 (B). Forty-eight hoursposttransfection, cells were treatedwith AZD6244 (2 mmol/L) for 90minutes. Cell lysates wereimmunoblotted to detect indicatedproteins. Cells expressing EGFRT669A were also treated with 50 ng/mL HRG ligand for 30 minutes toachieve maximal ERBB3phsophorylation. C, HCC827 cellswere infected with a control orshEGFR hairpin, followed byinfection with lentiviral vectorsexpressing GFP, T669 wild-typeEGFR (exon 19del), or EGFR T669A(exon 19del). After knockdown andpuroselection for 72 hours, cellsweretreatedwith AZD6244 (2mmol/L) for 6hours. Cell lysates wereimmunoblotted to detect theindicated proteins.

A B

p-ERBB3 (1289)

ERBB3

p-EGFR (1068)

EGFR

p-ERK

ERK

Actin

GFP WT 669A EGFR

WT ERBB3

- + - + - + - AZD6244

- - - - - - + HRG 50 ng/mL

p-ERBB3 (1289)

ERBB3

p-HER2 (1221/2)

HER2

p-ERK

ERK

Actin

GFP WT 677A HER2

WT ERBB3

- + - + - + AZD6244

C

p-ERBB3 (1289)

ERBB3

p-EGFR (1068)

EGFR

p-AKT (308)

p-AKT (473)

AKT

p-ERK

ERK

GFP WT 669A EGFR (exon19del)

Control shEGFR

- + - + - + - + AZD6244

Figure 7. Model of MEK inhibitor–induced feedback on ERBB receptorsignaling pathways.In untreated cells, EGFR isphosphorylated at T669 by MEK/ERK, which inhibits activation ofEGFR and ERBB3. In the presence ofAZD6244, ERK is inhibited and T669phosphorylation is blocked,increasing EGFR and ERBB3tyrosine phosphorylation andupregulating downstream signaling.

EGFR/EGFR EGFR/ERBB3

PI3KMEK

ERK AKT

EGFR/EGFR EGFR/ERBB3

AZD6244phospho-T

phospho-Y

Untreated MEK inhibitor

PI3KMEK

ERK AKT

Unbound RTKs






Furthermore, we show that although multiple mechanismsfor MAPK feedback regulation of AKT signaling have beenproposed, T669A mutation of EGFR is sufficient to blockMEK inhibitor-induced feedback activation of PI3K/AKT,suggesting that the feedback we describe herein is one of thedominant mechanisms regulating AKT activation in EGFR-and HER-driven cancers.

In addition to increased ERBB3 tyrosine phosphorylation,we also observe increased expression of total ERBB3 proteinafter MEK inhibition. This increase seems to be posttranscrip-tional as no change in ERBB3 mRNA levels was observed withAZD6244 (Supplementary Fig. S3). We were unable to defin-itively determine the mechanism for increased expression oftotal ERBB3. However, we observed that increased ERBB3expression was not solely a result of increased tyrosine phos-phorylation of ERBB3 (Fig. 4B). Interestingly, inhibition of ERK-mediated phosphorylation of the threonine juxtamembranedomain sites seems to be necessary for both increased phos-pho- and total ERBB3 levels. For example, expression of T669AEGFR in CHO-KI cells and HCC827 cells led to increased basalERBB3 expression and phosphorylation, which was not furtheraugmented by AZD6244 (Fig. 6). This suggests that theincreases in both phosho- and total ERBB3 are the result ofincreased dimer formation between EGFR and ERBB3, whichresults from loss of inhibitory threonine phosphorylationwithin the juxtamembrane domain of EGFR. Although webelieve that the data support such a model, it remains possiblethat phosphorylation of the EGFR juxtamembrane domainaffects tyrosine-phosphorylated and total ERBB3 levels via amechanism not linked to heterodimer formation.

Overall, this study suggests that combining MEK inhibi-tors with either ERBB or PI3K inhibitors may be effectivestrategies in the clinic. Although there are currently noapproved therapies targeting ERBB3, development of anti-ERBB3 antibodies is underway and our data suggests thepossible use of combining these antibodies with MEK inhi-bitors to block feedback activation of AKT in multiple cancermodels. Interestingly, we also observed feedback activationof ERBB3 after MEK inhibition in KRAS-mutant cancers thatexpress low basal levels of phospho-ERBB3 and therefore donot use ERBB3 to activate PI3K. This observation suggests

that MEK feedback on ERBB3 occurs in a range of cancers,regardless of dependence on ERBB signaling, and highlightsanother potential complication for patients treated exclu-sively with inhibitors of the RAF/MEK/ERK pathway. Forexample, in KRAS-mutant cancers that initially respond tosingle agent RAF/MEK inhibitors, chronic inhibition of thispathway may lead to persistent activation of EGFR or HER2.Therefore, these data suggest that activation of ERBB sig-naling may lead to resistance to single-agent RAF or MEKinhibitors.

Disclosure of Potential Conflicts of InterestJ.A. Engelman receives research support from AstraZeneca and Galaxo-

SmithKline and consults for AstraZeneca, GalaxoSmithKline, Novartis, andGenentech. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.B. Turke, J.A. EngelmanDevelopment of methodology: A.B. Turke, Y. Song, C. Costa, J.A. EngelmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.B. Turke, Y. Song, R. Cook, C.L. Arteaga, J.M. AsaraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.B. Turke, C. Costa, J.M. Asara, J.A. EngelmanWriting, review, and/or revision of themanuscript: A.B. Turke, Y. Song, C.L.Arteaga, J.M. Asara, J.A. EngelmanStudy supervision: J.A. Engelman

AcknowledgmentsThe authors thank Min Yuan and Susanne Breitkopf for help with mass

spectrometry, Mike Rothernberg and Cyril Benes for helpful discussions, andZachary Morris and Andrea McClatchey for assistance with biotin labelingexperiments.

Grant SupportThis study is supported by the NIH Gastrointestinal Cancer SPORE P50

CA127003, K08 grant CA120060-01, R01CA137008-01, R01CA140594,1U01CA141457-01, Lung SPORE P50CA090578, the V Foundation, AmericanCancer Society RSG-06-102-01-CCE, the Ellison Foundation Scholar Award, andthe American Lung Cancer Association Lung Cancer Discovery Award (all to J.A.Engelman); NIH 5P01CA120964-04 and NIH DF/HCC Cancer Center SupportGrant 5P30CA006516-46 (J.M. Asara); and R01CA80195 and Vanderbilt BreastCancer SPORE P50 CA98131 (C.L. Arteaga).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received November 19, 2011; revised March 23, 2012; accepted April 8, 2012;published OnlineFirst May 2, 2012.

References1. Engelman JA. Targeting PI3K signalling in cancer: opportunities,

challenges and limitations. Nat Rev Cancer 2009;9:550–62.2. Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer

Cell 2007;12:9–22.3. Montagut C, Settleman J. Targeting the RAF-MEK-ERK pathway in

cancer therapy. Cancer Lett 2009;283:125–34.4. Sun SY, Rosenberg LM,Wang X, Zhou Z, Yue P, Fu H, et al. Activation

of Akt and eIF4E survival pathways by rapamycin-mediated mamma-lian target of rapamycin inhibition. Cancer Res 2005;65:7052–8.

5. Carracedo A, Baselga J, Pandolfi PP. Deconstructing feedback-sig-naling networks to improve anticancer therapy with mTORC1 inhibi-tors. Cell Cycle 2008;7:3805–9.

6. O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, et al. mTORinhibition induces upstream receptor tyrosine kinase signaling andactivates Akt. Cancer Res 2006;66:1500–8.

7. Chandarlapaty S, Sawai A, Scaltriti M, Rodrik-Outmezguine V, Grbo-vic-Huezo O, Serra V, et al. AKT inhibition relieves feedback suppres-sion of receptor tyrosine kinase expression and activity. Cancer Cell2011;19:58–71.

8. Chakrabarty A, Sanchez V, Kuba MG, Rinehart C, Arteaga CL. BreastCancer Special Feature: feedback upregulation of HER3 (ErbB3)expression and activity attenuates antitumor effect of PI3K inhibitors.Proc Natl Acad Sci U S A 2012;109:2718–23.

9. Serra V, ScaltritiM, Prudkin L, EichhornPJ, IbrahimYH,ChandarlapatyS, et al. PI3K inhibition results in enhancedHER signaling and acquiredERK dependency in HER2-overexpressing breast cancer. Oncogene2011;30:2547–57.

10. Engelman JA, Settleman J. Acquired resistance to tyrosine kinaseinhibitors during cancer therapy. Curr Opin Genet Dev 2008;18:73–9.

Turke et al.





11. Faber AC, Li D, Song Y, Liang MC, Yeap BY, Bronson RT, et al.Differential induction of apoptosis in HER2 and EGFR addicted can-cers following PI3K inhibition. Proc Natl Acad Sci U S A 2009;106:19503–8.

12. Ono M, Hirata A, Kometani T, MiyagawaM, Ueda S, Kinoshita H, et al.Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancercell lines correlates with dependence on the epidermal growth factor(EGF) receptor/extracellular signal-regulated kinase 1/2 and EGFreceptor/Akt pathway for proliferation. Mol Cancer Ther 2004;3:465–72.

13. Hoeflich KP, O'Brien C, Boyd Z, Cavet G, Guerrero S, Jung K, et al. Invivo antitumor activity of MEK and phosphatidylinositol 3-kinaseinhibitors in basal-like breast cancer models. Clin Cancer Res 2009;15:4649–64.

14. MirzoevaOK, Das D, Heiser LM, Bhattacharya S, Siwak D, GendelmanR, et al. Basal subtypeandMAPK/ERKkinase (MEK)-phosphoinositide3-kinase feedback signaling determine susceptibility of breast cancercells to MEK inhibition. Cancer Res 2009;69:565–72.

15. YoonYK, KimHP,Han SW,HurHS,Oh doY, ImSA, et al. Combinationof EGFR and MEK1/2 inhibitor shows synergistic effects by suppres-sing EGFR/HER3-dependent AKT activation in human gastric cancercells. Mol Cancer Ther 2009;8:2526–36.

16. Lehr S, Kotzka J, Avci H, Sickmann A, Meyer HE, Herkner A, et al.Identification of major ERK-related phosphorylation sites in Gab1.Biochemistry 2004;43:12133–40.

17. Roshan B, Kjelsberg C, Spokes K, Eldred A, Crovello CS, Cantley LG.Activated ERK2 interacts with and phosphorylates the docking proteinGAB1. J Biol Chem 1999;274:36362–8.

18. Yu CF, Liu ZX, Cantley LG. ERK negatively regulates the epidermalgrowth factor-mediated interaction of Gab1 and the phosphatidylino-sitol 3-kinase. J Biol Chem 2002;277:19382–8.

19. EbiH,CorcoranRB,SinghA,ChenZ, SongY, Lifshits E, et al. Receptortyrosine kinases exert dominant control over PI3K signaling in humanKRAS mutant colorectal cancers. J Clin Invest 2011;121:4311–21.

20. Li X, Huang Y, Jiang J, Frank SJ. ERK-dependent threonine phos-phorylation of EGF receptor modulates receptor downregulation andsignaling. Cell Signal 2008;20:2145–55.

21. Red Brewer M, Choi SH, Alvarado D, Moravcevic K, Pozzi A, LemmonMA, et al. The juxtamembrane region of the EGF receptor functions asan activation domain. Mol Cell 2009;34:641–51.

22. Yeh TC, Marsh V, Bernat BA, Ballard J, Colwell H, Evans RJ, et al.Biological characterization of ARRY-142886 (AZD6244), a potent,highly selective mitogen-activated protein kinase kinase 1/2 inhibitor.Clin Cancer Res 2007;13:1576–83.

23. Rothenberg SM, Engelman JA, Le S, Riese DJ II, Haber DA, SettlemanJ. Modeling oncogene addiction using RNA interference. Proc NatlAcad Sci U S A 2008;105:12480–4.

24. Engelman JA, Janne PA, Mermel C, Pearlberg J, Mukohara T, Fleet C,et al. ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc Nat Acad Sci U S A2005;102:3788–93.

25. Engelman JA, Mukohara T, Zejnullahu K, Lifshits E, Borras AM, GaleCM, et al. Allelic dilution obscures detection of a biologically significantresistance mutation in EGFR-amplified lung cancer. J Clin Invest2006;116:2695–706.

26. Faber AC, Dufort FJ, Blair D, Wagner D, Roberts MF, Chiles TC.Inhibition of phosphatidylinositol 3-kinase-mediated glucose metab-olism coincides with resveratrol-induced cell cycle arrest in humandiffuse large B-cell lymphomas. Biochem Pharmacol 2006;72:1246–56.

27. Dibble CC, Asara JM, Manning BD. Characterization of Rictor phos-phorylation sites reveals direct regulation of mTOR complex 2 byS6K1. Mol Cell Biol 2009;29:5657–70.

28. EganDF, ShackelfordDB,MihaylovaMM,GelinoS, KohnzRA,MairW,et al. Phosphorylation of ULK1 (hATG1) by AMP-activated proteinkinase connects energy sensing to mitophagy. Science 2011;331:456–61.

29. Zheng B, Jeong JH, Asara JM, Yuan YY, Granter SR, Chin L, et al.Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 topromote melanoma cell proliferation. Mol Cell 2009;33:237–47.

30. Hsieh AC,MoasserMM. TargetingHER proteins in cancer therapy andthe role of the non-target HER3. Br J Cancer 2007;97:453–7.

31. Carraway KL III. E3 ubiquitin ligases in ErbB receptor quantity control.Semin Cell Dev Biol 2010;21:936–43.

32. Diamonti AJ, Guy PM, Ivanof C, Wong K, Sweeney C, Carraway KLIII. An RBCC protein implicated in maintenance of steady-stateneuregulin receptor levels. Proc Natl Acad Sci U S A 2002;99:2866–71.

33. Turke AB, Zejnullahu K, Wu YL, Song Y, Dias-Santagata D, Lifshits E,et al. Preexistence and clonal selection of MET amplification in EGFRmutant NSCLC. Cancer Cell 2010;17:77–88.

34. Hubbard SR. The juxtamembrane region of EGFR takes center stage.Cell 2009;137:1181–3.

35. Jura N, Endres NF, Engel K, Deindl S, Das R, Lamers MH, et al.Mechanism for activation of the EGF receptor catalytic domain by thejuxtamembrane segment. Cell 2009;137:1293–307.

36. Northwood IC, Gonzalez FA, Wartmann M, Raden DL, Davis RJ.Isolation and characterization of two growth factor-stimulated proteinkinases that phosphorylate the epidermal growth factor receptor atthreonine 669. J Biol Chem 1991;266:15266–76.

37. TakishimaK, Griswold-Prenner I, Ingebritsen T, RosnerMR. Epidermalgrowth factor (EGF) receptor T669 peptide kinase from 3T3-L1 cells isan EGF-stimulated "MAP" kinase. Proc Natl Acad Sci U S A 1991;88:2520–4.






2012;72:3228-3237. Published OnlineFirst May 2, 2012.Cancer Res Alexa B. Turke, Youngchul Song, Carlotta Costa, et al. Negative Feedback on ERBB ReceptorsMEK Inhibition Leads to PI3K/AKT Activation by Relieving a

Updated version

10.1158/0008-5472.CAN-11-3747doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2012/04/25/0008-5472.CAN-11-3747.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/72/13/3228.full#ref-list-1

This article cites 37 articles, 18 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/72/13/3228.full#related-urls

This article has been cited by 12 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/72/13/3228To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-11-3747

http://cancerres.aacrjournals.org/content/suppl/2012/04/25/0008-5472.CAN-11-3747.DC1

http://cancerres.aacrjournals.org/content/72/13/3228.full#ref-list-1

http://cancerres.aacrjournals.org/content/72/13/3228.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/72/13/3228


MEK Inhibition Leads to PI3K/AKT Activation by Relieving a ...Molecular and Cellular Pathobiology...

Documents

Transcript of MEK Inhibition Leads to PI3K/AKT Activation by Relieving a ...Molecular and Cellular Pathobiology...