differential effects of tyrosine kinase inhibitors on...

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Differential Effects of Tyrosine Kinase Inhibitors on Normal and Oncogenic EGFR Signaling and

Downstream Effectors

Youngjoo Kim1,4

, Mihaela Apetri1, BeiBei Luo

1, Jeffrey E. Settleman

2,3, and Karen S. Anderson

1*

1Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510

2Center for Molecular Therapeutics, Massachusetts General Hospital Cancer Center and Harvard Medical

School, Charlestown, MA 02129

3Current address: Genentech Inc. 1 DNA Way, South San Francisco, CA 94080

4Current address: Department of Chemistry and Physics, SUNY College at Old Westbury, 223 Store Hill Road,

Old Westbury, NY 11568

Running Title: Effect of gefitinib and erlotinib on normal and oncogenic EGFR signaling

Keywords: EGFR, autophosphorylation, gefitinib, erlotinib, signaling proteins

*Corresponding author: K. S. A. phone 203-785-4526, fax 203-785-7670, email: [email protected]

+This research was supported by National Institute of Health grants NCI CA127580 and CA125284 to K.S.A.

Disclosure: No conflicts of interest

on June 29, 2018. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 8, 2015; DOI: 10.1158/1541-7786.MCR-14-0326

mailto:[email protected]

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ABSTRACT

Constitutive activation of epidermal growth factor receptor (EGFR) due to overexpression or mutation in

tumor cells leads to dysregulated downstream cellular signaling pathways. Therefore, EGFR as well as its

downstream effectors have been identified as important therapeutic targets. The FDA approved small-molecule

inhibitors of EGFR, gefitinib (Iressa™) and erlotinib (Tarceva™), are clinically effective in a subset of non-

small cell lung cancer (NSCLC) patients whose tumors harbor activating mutations within the kinase domain of

EGFR. The current study examined effects of these drugs in 32D cells expressing native (WT) or oncogenic

(L858R) EGFR as well as in cancer cell lines A431 and H3255. Distinct patterns for gefitinib and erlotinib

inhibition of EGFR autophosphorylation at individual tyrosines were revealed for WT and L858R EGFR.

Phosphorylation of Y845 has been shown to be important in cancer cells and Y1045 phosphorylation is linked

to Cbl-mediated ubiquitination and degradation. Dramatic differences were observed by greater potency of

these drugs for inhibiting downstream effectors for L858R EGFR including Cbl and STAT5. Selective targeting

of Cbl, may play a role in oncogene addiction and effects on STAT5 identify features of signaling circuitry for

L858R EGFR that contribute to drug sensitivity and clinical efficacy. These data provide new understanding of

the EGFR signaling environment and suggest useful paradigms for predicting patient response to EGFR-

targeted therapy as well as combination treatments.

Implications: This study offers fundamental insights for understanding molecular mechanisms of drug

sensitivity on oncogenic forms of EGFR and downstream signaling components as well as considerations for

further drug optimization and design of combination therapy.




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Introduction

Cancer is the leading cause of death worldwide, and there are limited options for treatment, which

typically includes surgery, irradiation, and/or chemotherapy. Since the successful development of the first

kinase-targeted drug, imatinib (Gleevec), which inhibits the Abelson tyrosine kinase that promotes chronic

myelogenous leukemia (1), many drugs have been subsequently developed that target specific proteins that are

mutationally activated or overexpressed in various forms of cancers. Because EGFR is overexpressed or

constitutively active due to mutations in various cancers, it is an important target for drug development.

Erlotinib and gefitinib are two of the first FDA approved drugs specifically targeting EGFR in non-small cell

lung cancer (NSCLC). These compounds are small ATP mimetics that inhibit autophosphorylation of EGFR at

its cytoplasmic tyrosine residues by binding to the catalytic site. Notably, these two drugs are more effective in

patients with activating mutations in the kinase domain of EGFR. The most common activating EGFR

mutations are an in-frame deletion affecting exon 19 (delE746-A750) and a point mutation at residue 858 in

exon 21 (L858R)1 (2). The underlying mechanisms for enhanced potency are unclear. Previous structural

studies show similar inhibitor binding in the kinase domain for native (WT) and L858R mutant forms of EGFR

(3) and subsequent work has suggested altered ATP affinity, dimerization propensity, and conformational

enzyme dynamics as possible factors governing increased drug sensitivity (4, 5) (6).

Phosphorylated tyrosine residues in EGFR serve as specific recruitment sites for downstream signaling

molecules that are involved in various cellular processes such as proliferation, migration, and apoptosis (7).

Although studies have shown that different signaling molecules in pathways downstream of EGFR are activated

in many cancers, few global studies address linking autophosphorylation of a tyrosine residue in EGFR to the

activation of specific downstream signaling molecules.

Studies by Wu et al. show that many tyrosine residues in EGFR are differentially phosphorylated in lung

cancer compared to normal lung tissue and provide a phosphosignature that is strongly correlated with the lung

cancer phenotype (8). Similarly, Klammer et al. looked at the phosphoproteome profile from NSCLC cell lines

identifying predictive phosphosignatures that differ between sensitive and resistant cell lines (9). In another

study, Chen et al. report distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR

mutants compared to wild type EGFR (10). It was observed that gefitinib has variable growth-inhibitory effects

1 L858R is also known as L834R. Difference in numbering is based upon the 24 amino acid nuclear localization

sequence that is absent in the mature form of EGFR.




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in cells expressing different EGFR mutants. These studies show that various cell lines with distinct

phosphorylated EGFR due to mutations have specific phosphosignatures that respond differently to the drugs.

Therefore, correlating specific phosphosignatures to drug responsiveness may be a useful strategy for

individualized therapy.

Previously, our biochemical studies examined the temporal resolution of autophosphorylation for wild

type and oncogenic L858R EGFR using the recombinant cytoplasmic domain of EGFR expressed in Sf9 cells(5,

11). Differences in autophosphorylation kinetics and the unique signature patterns of drug sensitivity were

observed between wild type and L858R EGFR. With these biochemical studies as a foundation, we extended

our studies at a cellular level using 32D cells, a myeloid cell line lacking endogenous EGFR. Isogenic 32D cells

overexpressing either native (WT) or oncogenic L858R mutant forms of EGFR allowed the study of normal and

aberrant EGFR signaling and drug responsiveness without concern for cell line heterogeneity. Additional

studies examined WT and L858R mutant forms of EGFR in the setting of cancer cells. A431 is a human

epidermoid carcinoma cell line overexpressing EGFR and H3255 is a human lung cancer cell line expressing

L858R EGFR. These cell lines were included as part of an earlier study to understand the effects of the EGFR

antibody cetuximab in lung cancer cells and xenografts expressing oncogenic forms of EGFR (12). The current

study was designed to address the following mechanistic questions related to the clinical efficacy of gefitinib

and erlotinib: (1) Are differences in drug responsiveness observed in EGFR autophosphorylation patterns for

individual tyrosines in 32D cells expressing WT and L858R forms of EGFR? ; (2) Are some downstream

pathways more significant than others when comparing normal and oncogenic EGFR signaling?; and (3) Can

we identify key tyrosines in EGFR or downstream signaling molecules that may play prominent roles in

determining drug sensitivity in the context of oncogenic EGFR signaling?

The current study establishes that gefitinib and erlotinib have differential effects at a cellular level as

assessed by examining autophosphorylation of individual tyrosines in 32D cells expressing WT or L858R

mutant forms of EGFR, consistent with our previous biochemical studies. In addition, it was observed that there

were marked differences in drug sensitivity with respect to inhibition of downstream signaling proteins. By

examining normal and oncogenic EGFR signaling in 32D cells, it was found that both drugs significantly

impacted the activation of the Y845 residue in L858R EGFR compared to WT EGFR. Among downstream




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signaling proteins, STAT5 activation was substantially diminished by erlotinib (288-fold) and Cbl activation

was most affected by gefitinib (267-fold) in L858R EGFR signaling relative to WT EGFR signaling. Our results

suggest that L858R EGFR signaling may be mediated through activation of EGFR by autophosphorylation or

Src phosphorylation of Y845 followed by STAT5 activation. Inhibition of this pathway for L858R EGFR may

be linked to the effectiveness of gefitinib. Likewise, the potent inhibition of Cbl activation in L858R signaling

by erlotinib relative to WT EGFR may circumvent receptor degradation and contribute to an oncogene-addicted

cellular phenotype. This in-depth analysis of receptor activation, downstream signaling, and differential effects

of clinically important drugs aids in understanding mechanistic differences in normal and oncogenic EGFR

signaling. These major findings in 32D cell lines were well translated to cancer cell lines. These results provide

insights into the complexity of the EGFR signaling network in human tumors and pinpoint key features of

downstream signaling that may be essential for designing even more selective therapies.

Materials and Methods

Cell lines and culture conditions. The cell lines used in this study (32D, A431 and H3255 cells) were kindly

provided by Dr. Carlos Arteaga at Vanderbilt University School of Medicine (12) (13). The 32D and A431 cells

were originally obtained from American Tissue Culture. The NCI H3255 cells have been previously

characterized (14-16); they are heterozygous for the L858R missense mutation in exon 21 of the EGFR gene.

The IL-3-dependent hematopoietic 32D cell lines stably transfected with EGFR-WT and EGFR-L858R were

maintained in RPMI-1640 containing 10% fetal bovine serum (FBS) and 10% WEHI conditioned medium (as a

source of IL-3), and supplemented with antibiotics (penicillin/streptomycin 100U/mL). The human lung cancer

cell line NCI H3255 was grown in RPMI-1640 supplemented with 10% FBS plus antibiotics

(penicillin/streptomycin 100U/mL, gentamicin 50U/mL). The human epidermoid carcinoma cell line A431 was

maintained in Improved MEM Zn+2

Option (Richter’s Modification) supplemented with 10% FBS and

antibiotics (penicillin/streptomycin 100U/mL). All cells were grown at 37 °C in a humidified atmosphere with

5% CO2.

Reagents and Antibodies. Epidermal growth factor (EGF) was purchased from R&D Systems (Minneapolis,

MN). We utilized the following antibodies: anti-phospho-EGFR (to phosphotyrosines 1045, 1068, 1148, and

1173), total EGFR, Akt (#4691), phospho-Akt (Thr308, #9275), MAPK (#4695), phospho-MAPK (Thr202




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/Tyr204, #4370), PI3K (#11889), phospho-PI3K (Tyr458, #4228), PLC1 (#5690), phospho-PLC1 (Tyr783,

#2821), Src (#2123), phospho-Src (Tyr416, #2101), Stat3 (#4904), phospho-Stat3 (Tyr705, #9145), phospho-

Stat5 (Tyr694, #4322), Cbl (#2747), and Horseradish peroxidase (HRP)-linked rabbit IgG secondary antibody

from Cell Signaling (Danvers, MA); anti-phospho-EGFR (phosphotyrosines 845 and 992) from Millipore

(Billerica, MA); and Stat5 (sc-835), phospho-Cbl (Tyr700, sc-377571), and goat anti-mouse IgM-HRP (sc-

2064) from Santa Cruz Biotechnology (Dallas, TX).

Rapid cell stimulation with EGF. Before EGF stimulation, the 32D non-adherent cells grown to a density of

approximately 2x106 cells/ml were starved for 3 hours in serum-free RPMI-1640. After starvation, equal

volumes of the cell suspension and EGF ligand in RPMI-1640 medium (200 ng/ml) were mixed using a KinTek

RQF-3 rapid-quench instrument. The reactions were quenched at different time points with lysis buffer (50 mM

HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 10% glycerol, 1% triton X-100, 25 mM NaF,

protease inhibitors (Complete tablets, Roche Diagnostics) and 1mM sodium vanadate). The zero time point was

collected using RPMI-1640 media alone in the absence of EGF ligand and represented the unstimulated state.

Lysates were incubated on ice for 30 minutes, cleared by centrifugation at 13,000 g for 10 minutes and the

supernatants were collected for further analysis or stored at - 80°C.

Inhibition of EGFR Phosphorylation. 32D-EGFR WT and 32D-EGFR L858R stable cell lines were serum

starved as described previously (17). A431 and H3255 cells grown to 80% confluence were starved overnight in

serum-free medium. Before stimulation, cells were preincubated with a range of concentrations of gefitinib or

erlotinib (0-10M for WT and 0-0.1M for L858R EGFR) for 30 minutes and then stimulated with 100 ng/ml

EGF for 2 minutes. 32D cells were lysed directly with lysis buffer, while A431 and H3255 cells were washed

with ice-cold phosphate buffered saline (PBS) and scraped immediately after adding lysis buffer. All lysates

were cleared by centrifugation and supernatants were collected. Receptor phosphorylation was detected by

western blotting with specific phospho-EGFR antibodies.

Western Blot Analyses. Equal amounts of protein extracts (10 ng) were resolved by SDS–PAGE (BioRad,

7.5% Tris-HCl gels), and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford,




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MA, USA). The membrane was first incubated in blocking buffer (3% BSA in TBST- Tris Buffered Saline with

0.05% Tween20), then incubated with a primary antibody (dilution of antibodies were determined as described

previously (5)), washed, and blotted with secondary antibody conjugated with horseradish peroxidase (1:2000).

Blots were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) and

exposed to X-ray films. The level of receptor phosphorylation was evaluated from the western blotting using a

Molecular Imager FX. Standard curves for each antibody were generated to determine the dilution factor

necessary to obtain a signal that is linear in response to the amount of protein loaded. Adjusted antibody

dilution was as follows: 1:2000 for 845, 1:5000 for 992, 1:200 for 1045, 1:1000 for 1068, 1:250 for 1148,

1:5000 for 1173, and 1:2000 for EGFR. To estimate the rate of EGFR phosphorylation the intensity of bands

was plotted against increasing times and the data were fitted to equation 1. In order to determine the

concentration of kinase inhibitor (gefitinib or erlotinib) that resulted in 50% EGFR activity (IC50), the data were

fitted to the equation 2.

Intensity = Intensitymax*(1-exp (-time*rate of autophosphorylation) + constant (equation 1)

Percent activity = (activitymax*IC50) / (IC50 + [kinase inhibitor]) + constant (equation 2)

Results

Differential effect of gefitinib and erlotinib on autophosphorylation of EGFR

Previously we showed that the pattern of gefitinib sensitivity toward tyrosine residues determined at a

biochemical level is unique in WT and L858R EGFR (5, 11). Using recombinant EGFR expressed in Sf9 insect

cells, we observed that gefitinib was more potent for L858R EGFR than WT and the ranges of drug sensitivity

required to inhibit individual tyrosine residues were very different. To further examine drug sensitivity in a cell

culture context, we performed similar experiments using 32D cell lines. 32D cells are an IL-3-dependent

myeloid cell line and EGFR-expressing 32D cells become EGF-dependent in the absence of IL-3. Since there is

no endogenous expression of EGFR, 32D cell lines with stable expression of EGFR are widely used as an

isogenic model system for studying EGFR signaling. To obtain IC50 values of gefitinib and erlotinib for EGFR

inhibition, serum-starved 32D cells expressing WT or L858R EGFR were incubated with increasing




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concentrations of inhibitors for 30 min and then stimulated with EGF for 2 min as described in Materials and

Methods. EGF stimulation of 32D cell lines expressing WT and L858R mutant forms of EGFR and kinetic

analysis of EGFR phosphorylation established that a maximum level of EGFR activation was achieved after 2

min (data not shown). Levels of phosphorylation of individual tyrosine residues for WT and L858R mutant

EGFR in the presence of gefitinib or erlotinib were followed by western blot analysis using phospho-specific

EGFR antibodies (Figures 1 & 2). Similar to the biochemical data obtained previously, IC50 values for drugs

inhibiting the various tyrosine residues of EGFR varied widely at a cellular level (Supplementary Table 1). For

example, IC50 values for gefitinib for WT EGFR ranged from 0.019 to 0.083 M, and those for erlotinib ranged

from 0.015 to 0.079 M. L858R EGFR was more sensitive to both gefitinib and erlotinib than WT. In addition

to the overall differences in IC50 values, with erlotinib being more potent than gefitinib, there were differential

effects on the inhibition of tyrosine autophosphorylation in EGFR. For wild type EGFR, gefitinib was most

selective for Y1045 and Y992 (~0.02 M), followed by Y1173 (0.037 M), and Y1148, Y1068, and Y845

(0.07-0.083 M). Erlotinib inhibited phosphorylation of tyrosine residues in a similar order of potency as

gefitinib. For L858R EGFR, the pattern in drug sensitivity was altered relative to the WT. All tyrosine residues

except Y1068 were similarly sensitive to gefitinib with IC50 of 0.008-0.018 M versus 0.054 M for Y1068. As

with WT EGFR, erlotinib showed a similar pattern of sensitivity, but with greater potency. Interestingly,

gefitinib and erlotinib were most potent for inhibition of Y1045 in both wild type and L858R EGFR. This is

consistent with our observations at a biochemical level (5). Y1045 of EGFR is a docking site for Cbl proteins,

which are E3 ligases responsible for ubiquitination and degradation of EGFR (18, 19). Therefore, these results

suggest that drugs inhibit phosphorylation at Y1045 and prevent EGFR from ubiquitination.

To quantitatively assess differential drug effects on tyrosine residues of wild type and L858R EGFR, the

ratios of IC50 values (IC50 values of wild type EGFR/ IC50 values of L858R EGFR) were compared. These

values are represented as fold differences in parentheses in Figures 1C & 2C. Even though gefitinib and

erlotinib were more potent toward L858R EGFR than wild type EGFR in general (up to 6-fold for gefitinib and

up to 30-fold for erlotinib), the most striking difference observed was in potency of the two drugs on Y845




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phosphorylation. As shown in Figure 1C & 2C, the IC50 of gefitinib in inhibiting Y845 phosphorylation in

L858R EGFR was ~6-fold lower than that seen for wild type EGFR, and erlotinib had ~30-fold lower IC50 in

inhibiting Y845 of L858R EGFR than wild type. Y845 is located in the activation loop and has been suggested

to be phosphorylated by the Src kinase, which is required for EGF-induced tumorigenesis (20-22). The Y845

residue could also be autophosphorylated, but this is not required for EGFR activation, unlike for many other

receptor tyrosine kinases (22). These results suggest that the two EGFR inhibitors are more effective toward

L858R EGFR by specifically inhibiting phosphorylation of the Y845 residue, and that this effect is greater with

erlotinib compared to gefitinib.

Effect of gefitinib and erlotinib on phosphorylation of downstream signaling proteins

Autophosphorylation of EGFR leads to activation of downstream signaling (23). Gefitinib and erlotinib

have been shown to target EGFR by inhibiting autophosphorylation of EGFR tyrosine residues; however,

further effects of these drugs on downstream signaling proteins have not been previously studied in isogenic

cells in a comprehensive manner. Here we examined the effect of gefitinib and erlotinib on inhibition of

signaling proteins downstream of WT and L858R EGFR by measuring phosphorylation levels using western

blot analysis. Briefly, serum-starved 32D cells expressing WT or L858R EGFR were incubated with increasing

concentrations of drugs for 30 min and stimulated with EGF for 2 min. Phosphorylation levels of downstream

signaling proteins including Src, PI3K, Akt, STAT3, STAT5, ERK, PLC- and Cbl were followed by western

blot analysis using total and phosphospecific antibodies (Supplementary Figure 1). These particular signaling

proteins were selected because their overexpression and activation have been linked to EGFR activation and are

associated with cancer (24-27). Supplementary Table 2 summarizes IC50 values of gefitinib and erlotinib with

respect to eight downstream signaling proteins. These IC50 values do not represent direct inhibition of signaling

proteins by drugs, rather they examine how the upstream inhibition of EGFR phosphorylation by these drugs is

manifested downstream and whether inhibition of phosphorylation is amplified or dampened in the context of

WT and L858R EGFR signaling.




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As shown in Supplementary Table 2A, in 32 D cells expressing WT EGFR, the IC50 values of gefitinib for

downstream EGFR signaling proteins indicate that STAT3 and STAT5 are the most sensitive (0.017-0.068

M), followed by PLC- and PI3K (0.13-0.16 M), then Akt and ERK (0.92-1.08 M), and lastly, Cbl and Src

(3.74-4.12 M). Also in Supplementary Table 2A are results for the 32 D cells expressing L858R EGFR,

showing gefitinib was much more potent in exerting effects on downstream EGFR signaling proteins. Distinct

drug sensitivity patterns were observed. The IC50 values of gefitinib revealed that the downstream proteins most

sensitive were STAT5 and PLC- (0.0038-0.008 M), followed by ERK, Cbl, STAT3, and AKT (0.012-0.028

M); whereas, Src and PI3K were not inhibited by as much as the highest concentration of gefitinib tested (0.5

M). A comparison of fold-differences for WT/L858R cells are shown in the right hand column.

Similar to gefitinib, erlotinib was more potent toward L858R EGFR than wild type EGFR, with the

exception of Src and PI3K phosphorylation, as shown in Supplementary Table 2B. However, the pattern of

erlotinib inhibition of downstream signaling proteins was not the same as for gefitinib. In this case, the IC50

values for erlotinib toward WT EGFR signaling proteins showed that STAT3 was the most sensitive (0.0061

M), followed by Src and Cbl (0.042 and 0.05 M, respectively), and PI3K, Akt, STAT5, PLC-, and ERK

(0.46-0.84 M). For the L858R mutant EGFR signaling, the IC50 values of erlotinib for inhibition of

downstream phosphorylation of signaling proteins showed that STAT3 was the most sensitive (0.00087 M),

followed by Cbl, STAT5, and PLC- (0.0015-0.0048 M), and ERK and Akt (0.019 and 0.023 M,

respectively). Src and PI3K phosphorylation was not inhibited by as much as 0.5 M of erlotinib, as was seen

with gefitinib.

A summary of the ratio of IC50 values for each drug in the context of signaling proteins in WT and

L858R EGFR-expressing cells is shown as bar graphs in Figures 3A (gefitinib) & 3B (erlotinib). The data

reveal that these drugs exhibit distinct patterns of inhibition for downstream signaling pathways in WT and

L858R EGFR-expressing cells. Those values having >5-fold difference are shown. One of the most striking

results was the effect of gefitinib on Cbl phosphorylation, where the drug was 288-fold more potent on L858R




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EGFR relative to WT (Figure 3A). Cbl is an ubiquitin ligase and is responsible for ubiquitination and ultimate

targeting of EGFR for receptor degradation. On the other hand, erlotinib showed the greatest fold difference in

IC50 values between wild type and L858R EGFR expressing cells in the context of inhibition of downstream

signaling protein STAT which in this case was 267-fold (Figure 3B). These results suggest that the downstream

pathway effects of gefitinib and erlotinib are achieved in unique ways despite their similar chemical properties

and direct inhibition on EGFR.

To examine whether the effect of drugs on phosphorylation of downstream EGFR signaling proteins

seen in 32D cells is also observed in cancer cell lines, these studies were extended to evaluate A431 and H3255

cell lines. The A431 cancer cells overexpress WT EGFR while the H3255 cells overexpress the missense

oncogenic L858R EGFR. The data for the effects of gefitinib and erlotinib on A431 and H3255 are found in

Supplementary Table 3A and B and Supplementary Figure 2A and B, respectively. Figure 4 summarizes in bar

graphs the fold-difference in the ratio of IC50 values for WT (A431) and L858R (H3255) inhibition of

downstream EGFR proteins for gefitinib (Figure 4A) and erlotinib (Figure 4B). Those values showing >5-fold

inhibition are shown. Consistent with the results in 32D cells, in cancer cells expressing WT or L858R EGFR,

the largest effect on downstream EGFR signaling observed for gefitinib was on Cbl (100-fold) as shown in

Figure 4A. Also in accord with 32D cells as illustrated in Figure 4B, for erlotinib the most notable effect on

EGFR mediated downstream signaling partners for WT versus L858R was STAT5 (404-fold).

DISCUSSION

Cellular signaling via EGFR is initiated by ligand-stimulated autophosphorylation of key tyrosine residues of

EGFR that have been shown to recruit a host of downstream signaling proteins leading to activation of various

cellular pathways responsible for controlling growth and survival (28). Our previous study with FGFR1

(fibroblast growth factor receptor 1) have shown that the order of autophosphorylation is important in providing

both temporal and spatial resolution to receptor signaling and this order is perturbed in an oncogenic form of

FGFR1 (29, 30). Therefore, the pattern and timing of phosphorylation events on EGFR may be important for

proper orchestration of downstream partner recruitment and activation of relevant pathways in a defined manner




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for appropriate regulation of cell growth. Identifying specific phosphosignatures of global EGFR signaling may

offer insights that may be useful for predicting patient response to EGFR-targeted therapy as well as

combination treatments. Our previous biochemical studies using a recombinant form of the cytoplasmic domain

for EGFR expressed in Sf9 cells have shown that there is a unique signature pattern of drug sensitivity between

WT and L858R EGFR, indicating that the activation of specific tyrosine residues determines drug sensitivity

(5). The current study was designed to extend those findings in a cellular context to determine how inhibition

of EGFR autophosphorylation by gefinitib and erlotinib may differentially affect specific downstream signaling

proteins. EGFR autophosphorylation patterns may emerge that predict drug sensitivity and explain the

effectiveness of gefitinib and erlotinb in cells expressing L858R oncogenic form of EGFR relative to WT.

Downstream signaling partners and pathways may be identified that are also incongruently influenced in

oncogenic signaling and define essential features of drug efficacy.

Similar to our findings at the biochemical level, tyrosine 1045 within the C-terminal tail of EGFR was

identified as being one of the most sensitive to drug inhibition (Supplementary Table 1, Figures 1 & 2) (5).

Phosphorylation of Y1045 has been established as a mechanism to recruit Cbl, an ubiquitin ligase, for EGFR

ubiquitination followed by degradation in the lysosome (31, 32). In another study by Gui et al., hypo-

phosphorylation at Y1045 was shown to cause impaired degradation and enhanced recycling of EGFR (33). Our

results with gefitinib suggest that this drug may prevent the oncogenic L858R form of EGFR from degradation

by inhibiting phosphorylation at Y1045. If this mutant form of EGFR is less susceptible to degradation, this in

turn might render cell growth more dependent on EGFR activation, thereby enhancing oncogene “addiction”

(34). Accordingly, the mode of action for gefitinib in cells expressing L858R EGFR may in part be linked to

inhibition of Y1045 and subsequent prevention of Cbl activation, since Cbl is downstream of Y1045. However,

relative to L858R-expressing cells, gefitinib is a less potent inhibitor of Cbl phosphorylation in cells expressing

WT EGFR (Figure 3A and Figure 4A). In this case, Cbl might be activated through a different pathway. These

observations may explain the molecular features of inhibiting key tyrosines on EGFR and downstream effectors




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responsible for the clinical effectiveness of gefitinib in treating patients having EGFR mutations. Recent studies

have suggested that Cbl inhibitors may be effective in some cancers (35).

Our findings with erlotinib imply that additional mechanisms may by operative in patients with tumors

harboring EGFR mutations such as L858R. The most significant difference in terms of EGFR activation in

comparing cells expressing wild type and oncogenic EGFR was found to be erlotinib sensitivity toward

inhibition of phosphorylation of Y845 in EGFR, with an observed 30-fold difference in IC50 WT/ IC50 L858R as

shown in Supplementary Table 1B. Further examination of downstream signaling in the presence of erlotinib

revealed differences in the ratios for IC50 WT/ IC50 L858R for STAT5 and PLC- phosphorylation, with the

largest difference being STAT5 (Figure 3B). This large difference implies that effectiveness of erlotinib in

patients with EGFR mutation may be largely driven by inactivation of the STAT5 pathway perhaps in concert

with PLCγ1. Similar results were obtained with cancer cell lines in which a 404-fold difference in erlotinib

sensitivity was noted for STAT5 (Figure 4B).

The differential erlotinib activity against Y845 suggests that L858R EGFR might also be more

dependent on Y845 activation relative to wild type EGFR. Therefore, inhibiting Y845 phosphorylation by

erlotinib might provide a further explanation for the effectiveness in the context of EGFR L858R mutation.

Phosphorylation of Y845 has shown to be ligand-independent and associated with STAT-dependent gene

expression (36). Upon phosphorylation, the Y845 residue interacts with Src through an SH2 domain that in turn

activates STAT5 signaling. Y845 phosphorylation could be indirectly inhibited due to Src inhibition by drugs.

However, as shown in Supplementary Table 2 and 3, Src activity is not substantially changed by the two drugs.

Therefore, it seems plausible that Y845 phosphorylation is directly inhibited by drugs rather than via Src

inhibition. The lack of Src inhibition by gefitinib suggests that L858R activation is Src-independent.

Interestingly, studies done by Fu, Y-N et al. showed that phosphorylation of Y845 is important for constitutive

activation of L858R EGFR. They also found that L858R EGFR is more sensitive to PP2, a Src inhibitor,

compared to WT EGFR (36). This implies that L858R EGFR utilizes Y845 phosphorylation as an activation

mechanism, and that Src inhibition by PP2 would preclude Y845 phosphorylation from exhibiting enhanced

sensitivity toward PP2. Other work by Yang et al., suggests that mutation in the kinase domain of EGFR alone

is sufficient for phosphorylation of Y845 and that Src kinase activity is dispensable (36). An additional study by




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Chung et al. showed that NSCLC-associated mutant forms of EGFR displayed enhanced association with Src

(37). Activation of Y845 in L858R EGFR was also observed by Sordella et al (38).

Whether or not Y845 phosphorylation is Src-dependent, L858R EGFR signaling seems to involve Y845

activation. The results described in the current study of gefitinib and erlotinib are in accord with a similar

mechanism in which L858R EGFR becomes constitutively active due to the phosphorylation of the Y845

residue. Therefore, the inhibitory drugs are more effective toward L858R EGFR by specifically targeting Y845.

Another important consequence of Src-mediated phosphorylation of Y845 in EGFR signaling is the

activation of STAT5 (39, 40). Phosphorylation of Y699 within STAT5b is responsible for mediating EGF-

induced DNA synthesis. Interestingly, a connection in EGFR signaling between EGFR Y845 and STAT5 is

represented in Figures 3B and 4B and Supplementary Table 2B and 3B. In the case of erlotinib, the level of

phosphorylation for STAT5 was much lower in the presence of drug suggesting a possible link between Y845

phosphorylation and STAT5 activation. Src phosphorylation of EGFR on Y845 has a major role in regulation of

cellular functions, as described in reports by Sato et al (41). Src is negatively regulated by phosphorylation and

its signal-dependent transient activation contributes to cellular responses such as proliferation and

differentiation. Although Y845 is dispensable for cellular functions regulated by EGFR, trans-phosphorylation

of Y845 by Src plays an important role in several aspects of cellular functions. Thus, it was reported by Gotoh

et al. (42) that the Y845F mutant of EGFR was able to transform NIH3T3 cells. STAT is a possible mediator of

the Y845 phosphorylation-dependent synergy between EGFR and Src. In breast cancer cells, STAT5b was

identified as a major tyrosine-phosphorylated protein and expression of the Y845F mutant of EGFR had a

negative effect on activation of STAT5b. Moreover, activated EGFR-STAT3 signaling promotes tumor survival

in vivo in NSCLC (43).

Our study suggests that the efficacy of erlotinib in L858R EGFR-expressing 32D cells is a result of drug

effects on Y845 and STAT5 phosphorylation. Sordella et al. reported that activating mutations in EGFR

selectively activate Akt and STAT pathways that are important in lung cancer cell survival by activating an

anti-apoptotic pathways and gefitinib inhibits this survival pathway in EGFR-dependent cells. (38). Other

studies support a role for Src family kinases to link signals from growth factors to downstream effector

pathways (44, 45). An understanding of these phosphorylation events in signal propagation enables prediction

of drug sensitivity to small molecule inhibitors such as gefitinib and erlotinib. Song et al. showed that a Src




15

kinase inhibitor, dasatinib, selectively induces apoptosis in lung cancer cells dependent on EGFR signaling for

survival (46) suggesting that Src is in fact the main player in EGFR signaling. Our studies further validate the

importance of Src in linking EGFR signaling to activation of the STAT pathway via phosphorylation of tyrosine

845 in EGFR, and suggest that drugs such as gefitinib and erlotinib are much more effective in the context of

EGFR activating mutations due to their specific inhibition of phosphorylation of tyrosine residue 845, leading to

apoptosis. Activation of Src and STAT5 has also been reported in squamous cell carcinoma of the head and

neck (SCCHN) (47, 48). Koppikar et al. showed that combined treatment with Src inhibitor AZD0530 and

gefitinib led to greater inhibition of SCCHN compared with single inhibitor treatment (49). In another study,

Koppikar et al. presented that STAT5 activation contributes to the resistance of SCCHN cells to the EGFR

tyrosine kinase receptor, erlotinib (50). Taken together with our studies, combination therapy using inhibitors of

both STAT5 and EGFR would be more effective treatment in lung cancer patients. Many inhibitors targeting

STAT pathway in various cancers has been developed (51).

In summary, we have examined the effect of two FDA approved TKIs, gefitinib and erlotinib on

inhibition of EGFR and its downstream signaling proteins. The IC50 values for gefitinib and erlotinib inhibition

with respect to key tyrosine residues in EGFR and essential downstream signaling proteins were determined for

normal and oncogenic EGFR signaling. The most significant difference in terms of EGFR activation was found

to be erlotinib activity toward tyrosine 845 in L858R EGFR signaling, which exhibited ~30-fold lower IC50

value than normal WT signaling. For downstream signaling proteins, a 288-fold difference in IC50 WT/ IC50

L858R was observed for Cbl inhibition following gefitinib in 32D cells. Erlotinib showed a 267 difference in

IC50 WT/ IC50 L858R for STAT5 phosphorylation in 32D cells. Similarly, in comparing A431 cells and H3255,

there was 100-fold difference in IC50 WT/ IC50 L858R for Cbl inhibition following gefitinib and 404-fold

difference in IC50 WT/ IC50 L858R for STAT5 inhibition following erlotinib. Figure 5 provides a diagrammatic

representation of gefitinib and erlotinib effects on EGFR downstream signaling pathways. These studies

highlight the importance of Y1045 in EGFR and downstream signaling protein Cbl for the efficacy of gefitinib

on L858R EGFR. In addition, they suggest that constitutive activation of L858R EGFR is mediated through

phosphorylation of Y845 followed by activation of the STAT5 downstream pathway, and erlotinib seems to

specifically target these pathways in the context of oncogenic mutant forms of EGFR. This information

provides a more detailed understanding of the effectiveness of gefitinib and erlotinib. Moreover, this in-depth




16

analysis may point to new avenues to explore for designing better drugs and combination therapy targeting not

only EGFR but also its downstream signaling proteins when treating various forms of cancer.

Acknowledgement

We thank Dr. Carlos Arteaga at Vanderbilt University School of Medicine for kindly providing cell lines and

Dr. Christal Sohl for helpful comments on the manuscript.




17

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24

Figure Legends.

Figure 1. Effect of gefitinib on (A) WT-and (B) L858R-EGFR autophosphorylation in 32D cells. (C) Bar graph

representation of IC50 values of gefitinib for wild type and L858R EGFR in 32D cells. Parentheses indicate the

ratio of IC50 values of gefitinib for wild type relative to those of L858R EGFR. After serum starvation, cells

were preincubated with the indicated concentrations of gefitinib for 30 minutes and then stimulated with 100

ng/ml EGF for 2 minutes. Cell lysates were run on SDS-PAGE and receptor phosphorylation at different

tyrosine residues was detected by western blotting with specific phospho-EGFR antibodies.

Figure 2. Effect of erlotinib on (A) WT- and (B) L858R-EGFR autophosphorylation in 32D cells. (C) Bar graph

representation of IC50 values of erlotinib for wild type and L858R EGFR in 32D cells. Parentheses indicate the

ratio of IC50 values of erlotinib for wild type to that of L858R EGFR. After serum starvation, cells were

preincubated with the indicated concentrations of erlotinib for 30 minutes and then stimulated with 100 ng/ml

EGF for 2 minutes. Cell lysates were run on SDS-PAGE and receptor phosphorylation at different tyrosine

residues was detected by western blotting with specific phospho-EGFR antibodies.

Figure 3. (A) Bar graph representation of fold-difference in ratio of IC50 values of gefitinib inhibition of

downstream signaling partners for wild type and L858R EGFR in 32D cells. (B) Bar graph representation of

fold-difference in ratio of IC50 values of erlotinib inhibition of downstream signaling partners for wild type and

L858R EGFR in 32D cells. In each case, only those values having >5-fold difference are shown. In each case,

after serum starvation, cells were preincubated with the indicated concentrations of gefitinib or erlotinib for 30

minutes and then stimulated with 100 ng/ml EGF for 2 minutes. Cell lysates were run on SDS-PAGE and

various EGFR signaling proteins were detected by western blotting with total and phospho-antibodies.

Figure 4. (A) Bar graph representation of fold-difference for ratio of IC50 values of gefitinib for wild type versus

L858R EGFR in A431 and H3255 cell lines. (B) Bar graph representation of fold-difference for ratio of IC50

values of erlotinib for wild type versus L858R EGFR in A431 and H3255 cells lines. In each case, after serum

starvation, cells were preincubated with the indicated concentrations of gefitinib or erlotinib for 30 minutes and




25

then stimulated with 100 ng/ml EGF for 2 minutes. Cell lysates were run on SDS-PAGE and various EGFR

signaling proteins were detected by western blotting with total and phospho-antibodies.

Figure 5. Diagram of EGFR signaling pathway showing impact of gefitinib and erlotinib on EGFR downstream

signaling proteins.




Published OnlineFirst January 8, 2015.Mol Cancer Res Youngjoo Kim, Mihaela Apetri, Beibei Luo, et al. Oncogenic EGFR Signaling and Downstream EffectorsDifferential Effects of Tyrosine Kinase Inhibitors on Normal and

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