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RESEARCH ARTICLE

Multivariate signaling regulation by SHP2 differentially controlsproliferation and therapeutic response in glioma cells

Christopher M. Furcht1,*, Janine M. Buonato1,*, Nicolas Skuli2,3, Lijoy K. Mathew2,3, Andres R. Munoz Rojas4,M. Celeste Simon2,3 and Matthew J. Lazzara1,4,5,`

ABSTRACT

Information from multiple signaling axes is integrated in the

determination of cellular phenotypes. Here, we demonstrate this

aspect of cellular decision making in glioblastoma multiforme (GBM)

cells by investigating the multivariate signaling regulatory functions

of the protein tyrosine phosphatase SHP2 (also known as

PTPN11). Specifically, we demonstrate that the ability of SHP2 to

simultaneously drive ERK1/2 and antagonize STAT3 pathway

activities produces qualitatively different effects on the phenotypes

of proliferation and resistance to EGFR and c-MET co-inhibition.

Whereas the ERK1/2 and STAT3 pathways independently promote

proliferation and resistance to EGFR and c-MET co-inhibition,

SHP2-driven ERK1/2 activity is dominant in driving cellular

proliferation and SHP2-mediated antagonism of STAT3

phosphorylation prevails in the promotion of GBM cell death in

response to EGFR and c-MET co-inhibition. Interestingly, the

extent of these SHP2 signaling regulatory functions is diminished

in glioblastoma cells that express sufficiently high levels of

the EGFR variant III (EGFRvIII) mutant, which is commonly

expressed in GBM. In cells and tumors that express EGFRvIII,

SHP2 also antagonizes the phosphorylation of EGFRvIII and c-MET

and drives expression of HIF-1a and HIF-2a, adding complexity to

the evolving understanding of the regulatory functions of SHP2 in

GBM.

KEY WORDS: Signal transducer and activator of transcription-3

(STAT3), Extracellular signal-regulated kinase-1/2 (ERK1/2),

Epidermal growth factor receptor (EGFR), HGF receptor (c-MET),

Hypoxia inducible factors, Gefitinib

INTRODUCTIONCells integrate information from multiple signaling pathways toexecute decision-making processes (Janes et al., 2005; Wolf-Yadlin et al., 2006; Kumar et al., 2007; Lazzara and Lauffenburger,

2009). Whereas some signaling pathway intermediates actpredominantly in one pathway, others exert substantial effects inmultiple pathways, thus expanding their ability to control cell fate

determination. One such protein is SH2 domain-containing

phosphatase-2 (SHP2, also known as PTPN11), which plays key

roles in development, homeostatic maintenance and disease. Here,

we investigate the ability of SHP2 to simultaneously regulate

the extracellular signal-regulated kinase-1/2 (ERK1/2, hereafter

referred to as ERK) and signal transducer and activator of

transcription-3 (STAT3) pathways, as well as other signaling

events that we identify as SHP2-regulated for the first time, and the

net effect of this regulation on cellular proliferation and response to

co-inhibition of EGFR and the HGF receptor c-MET.

SHP2 was the first phosphatase to be identified as a proto-

oncogene (Chan and Feng, 2007; Chan et al., 2008) and is

primarily regarded as a mediator of proliferative and pro-survival

signaling. Indeed, the best-studied signaling role of SHP2 is to

promote ERK activity (Neel et al., 2003). The catalytic activity of

SHP2, which is required for this function, is promoted through

engagement of its N-terminal SH2 domains by phosphotyrosines

on various receptor tyrosine kinases or adapter proteins, such as

GRB2-associated binding protein-1 (GAB1) (Gu and Neel, 2003;

Neel et al., 2003). SHP2 can also negatively regulate STAT3

activation downstream of the interleukin-6 receptor (Schmitz

et al., 2000), and one recent study even described a ‘tumor-

suppressor’ role for SHP2 in hepatocellular carcinoma through its

regulation of STAT3 (Bard-Chapeau et al., 2011). SHP2 can also

positively or negatively impact AKT pathway activity (Wu et al.,

2001; Zhang et al., 2002). Through these signaling regulatory

functions, the magnitude of which may depend on cell type or

disease context, SHP2 is able to control cellular phenotypes

including proliferation (Cai et al., 2002; Furcht et al., 2012),

oncogenic transformation (Zhan et al., 2009), tumor progression

(Aceto et al., 2012), response to therapeutics (Furcht et al., 2012)

and senescence (Sturla et al., 2011).

A specific setting of interest where SHP2 influences multiple

complex phenotypes is glioblastoma multiforme (GBM), the most

common and lethal form of adult brain cancer (Zhang et al.,

2012). One study described the ability of SHP2 to suppress

cellular senescence in the GBM cell lines U87MG and A172, and

reported simultaneous SHP2-mediated activation of ERK and

inhibition of STAT3, although no causal relationships between

ERK or STAT3 signaling and the senescence phenotype were

established (Sturla et al., 2011). SHP2 function has also been

linked to tumorigenicity of GBM cells that express the EGFR

variant III (EGFRvIII), a mutant prevalent in GBM (Zhan et al.,

2009). Of course, the role of ERK and STAT3 in promoting

proliferation and survival across cancer types has been described

in depth (Hodge et al., 2005; Roberts and Der, 2007). For

example, in GBM cells, ERK activity promotes resistance to the

chemotherapy drug cisplatin (Zhan and O’Rourke, 2004), and

STAT3 is an important regulator of proliferation that has been

1Department of Chemical and Biomolecular Engineering, University ofPennsylvania, Philadelphia, PA 19104, USA. 2Abramson Family Cancer ResearchInstitute, University of Pennsylvania, Philadelphia, PA 19104, USA. 3HowardHughes Medical Institute, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA 19104, USA. 4Department of Bioengineering,University of Pennsylvania, Philadelphia, PA 19104, USA. 5Biochemistry andMolecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia,PA 19104, USA.*These authors contributed equally to this work

`Author for correspondence (mlazzara@seas.upenn.edu)

Received 11 March 2014; Accepted 4 June 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3555–3567 doi:10.1242/jcs.150862

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recognized as a potential therapeutic target (Lo et al., 2008; de laIglesia et al., 2009). Because SHP2 regulates the ERK and

STAT3 pathways in qualitatively different ways, and the ERK andSTAT3 signaling pathways promote qualitatively similar effectsacross multiple phenotypes, how the multivariate signaling roles ofSHP2 are integrated to determine phenotype in GBM cells is

unclear. Gaining a more complete understanding might help toaddress a number of outstanding issues, such as how GBMresistance to targeted inhibitors can be overcome (Sathornsumetee

et al., 2007; Argyriou and Kalofonos, 2009; De Witt Hamer, 2010)and the potential efficacy of targeting SHP2 in glioblastoma.

Here, we evaluate the effects of the signaling roles of SHP2 on

GBM cell proliferation and resistance to inhibitors of EGFR and c-MET, oncogenic receptors that drive GBM progression andchemoresistance. In a panel of GBM cell lines, SHP2 depletion

reduced cellular proliferation but, surprisingly, also promotedresistance to EGFR and c-MET co-inhibition. These results appearto derive from the ability of SHP2 to drive ERK and antagonizeSTAT3 pathway activities in the panel of cell lines and the

differential abilities of those pathways to control differentphenotypes. That is, even though ERK and STAT3 both promoteproliferation and survival, SHP2-mediated ERK activity is

dominant in determining cellular proliferation rates, whereassuppression of STAT3 phosphorylation through SHP2 exerts the

dominant effect in determining response to EGFR and c-MET co-inhibition. Interestingly, the ability of SHP2 to regulate these

pathways was greatly diminished in cells that expressedsufficiently high levels of EGFRvIII, where SHP2 was basallysequestered with the receptor. We further found that SHP2negatively regulates EGFRvIII and c-MET phosphorylation and

drives expression of hypoxia-inducible factors 1 and 2 alpha (HIF-1a and HIF-2a, respectively; hereafter referred to as HIF-1/2a) incultured cells and tumor xenografts. These results expand our

understanding of SHP2 as a multivariate regulator of signaling andGBM cell phenotype and raise additional questions about howSHP2 function may be perturbed in different GBM contexts.

RESULTSSHP2 depletion differentially impacts key GBM cellphenotypes and associated signaling pathwaysUsing the GBM cell lines U87MG, LN18, T98G and U118MG,we first evaluated the effect of small hairpin RNA (shRNA)-mediated SHP2 knockdown on cellular proliferation. As expected

based on reports that used other glioblastoma cells (Zhan et al.,2009) and several other cell settings, SHP2 knockdown reducedcellular proliferation rates in all four cell lines (Fig. 1A).

Interestingly, SHP2 knockdown also promoted cell survival inresponse to co-inhibition of EGFR and c-MET using the

Fig. 1. SHP2 knockdown differentially impacts GBM cell proliferation and survival. (A) U87MG, LN18, T98G and U118MG cells that express controlor SHP2-targeting shRNA were seeded at 100,000 cells/well, and cells were counted 72 h later. Counts are represented as the mean 6 s.e.m. (n53); *P,0.05.(B) The indicated cell lines were co-treated with 20 mM gefitinib + 1 mM PHA665752 (G+P) or with DMSO as a control (DMSO). After 72 h, the percentage of TO-PRO-3-positive cells was measured by flow cytometry (n53); *P,0.05. (C) The indicated cell lines expressing control or SHP2-targeting shRNA weremaintained in complete medium. Lysates were analyzed by western blotting using antibodies against the indicated proteins. Densitometry data are representedas the mean 6 s.e.m. (n53); *P,0.05.

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inhibitors gefitinib and PHA665752, respectively (Fig. 1B).Thus, in response to SHP2 knockdown, cells were generally

less proliferative but significantly more resistant to EGFR and c-MET co-inhibition. The latter effect was unexpected, givenprevious findings that SHP2 knockdown enhances cell death inresponse to EGFR inhibition in non-small cell lung cancer

(NSCLC) cells (Furcht et al., 2012) and that SHP2 antagonizesp73-dependent apoptosis (Amin et al., 2007). Western blot analysisrevealed that SHP2 knockdown was accompanied by a

concomitant decrease in ERK phosphorylation and increasein STAT3 phosphorylation (Fig. 1C), which could explain whyproliferation was impeded while survival in response to EGFR and

c-MET co-inhibition was enhanced. That is, ERK activity mightcontribute more substantially to determining proliferation rates,and STAT3 activity might contribute more substantially to survival

in response to EGFR and c-MET co-inhibition.To explore this idea further, we used the data shown in Fig. 1A-

C to assign quantitative values to the individual contributions ofSHP2-controlled ERK and STAT3 pathway activities towards

cellular proliferation and survival. We assumed that thequantitative measurement of a particular phenotype Xi under aparticular cellular condition i (in this case, control or SHP2

knockdown) can be described as a linear combination of thephosphorylation levels of ERK and STAT3 (pE,i and pS,i,respectively), where the specific contribution of each pathway to

Xi is determined by the product of a weighting coefficient for ERKor STAT3 (wE or wS, respectively) and the phosphorylation level ofthe protein. With these assumptions, Xi is defined as:

Xi~wE(pE,i)zwS(pS,i):

To evaluate pathway contributions to survival in response totherapeutics, the percentage of dead cells shown in Fig. 1B wassubtracted from 100% to determine the percentage of surviving

cells. Western blot signals of phosphorylated ERK and STAT3were normalized to the corresponding signals of total protein, asshown in Fig. 1C. Finally, phosphorylation and phenotype datawere normalized to values obtained from cells treated with control

shRNA for each cell line, which led to wE and wS summing to onewhen the equation above was evaluated for the control condition.Performing the analysis for the proliferation phenotype for each

cell line and averaging the data, we found average wE and wS

values of 0.77 and 0.23, respectively. For cell survival in responseto EGFR and c-MET co-inhibition, we found average wE and wS

values of 20.14 and 1.14, respectively. These results suggest thatERK and STAT3 play dominant roles in proliferation and survivalresponse, respectively. We note that a negative value for wE in the

survival analysis might seem to suggest that ERK activitysomehow negatively contributes to cell survival, but this is notthe case. Rather, this result arises owing to the form of our modelfor Xi, which produces a wE ,0 whenever the fold-increase in

survival exceeds the fold-increase in STAT3 phosphorylation, andthe fold-increase in ERK phosphorylation does not exceed that forSTAT3 phosphorylation, which is the case for three of the four cell

lines analyzed.

ERK and STAT3 inhibition further suggests differentialpathway control of proliferation and survival in GBM cellsWe next used the ERK and STAT3 inhibitors CI-1040 and Stattic,respectively, to confirm independently the relative contributions

of ERK and STAT3 to cell phenotypes. Cellular proliferationwas reduced with either ERK or STAT3 pathway inhibition

(Fig. 2A,B; supplementary material Fig. S1A). Note that theincomplete inhibition of STAT3 phosphorylated at residue Y705

(37% reduction) observed in Fig. 2B resulted from our selectionof a STAT3 inhibitor concentration that was low enough toproduce relatively low levels of cell death as a single agent acrossthe panel of cell lines. Using a lower concentration of gefitinib

than the one used in the experiments shown in Fig. 1B to reducebaseline cell death, we also found that ERK or STAT3 inhibitionpromoted cell death in response to EGFR and c-MET co-

inhibition (Fig. 2C). With the exception of U118MG cells whereStattic produced a substantial amount of cell death by itself, theeffect of ERK inhibition on proliferation was generally greater

than that of STAT3 inhibition. By contrast, the effect of STAT3inhibition on cell death in response to gefitinib and PHA665752was larger than that of ERK inhibition. Given that the same

concentrations of CI-1040 and Stattic were used in theexperiments shown in Fig. 2A,C, we interpret these data asindicating that both the ERK and STAT3 pathways participatein the regulation of cellular proliferation and survival, but

confirming the weighting coefficient analysis conclusion thatERK is the stronger determinant of proliferation and STAT3 thestronger determinant of survival in response to EGFR and c-MET

co-inhibition. This suggests that the elevated levels ofphosphorylated STAT3 observed with SHP2 knockdownpromoted resistance to EGFR and c-MET co-inhibition

despite the impairment of ERK activity. To confirm this, wedemonstrated that combining Stattic with the concentrations ofgefitinib and PHA665752 used as shown in Fig. 1B increased the

cell death response of cells in which SHP2 was knocked down(Fig. 2D).

We note as well that, in some cell lines, increases in STAT3Y705 phosphorylation might involve a mechanism wherein

ERK negatively regulates STAT3 Y705 phosphorylation byphosphorylating STAT3 S727 (Chung et al., 1997). Evidencefor this potential connectivity between ERK and STAT3 is

provided by our finding that MEK inhibition promoted STAT3phosphorylation in some cell lines (supplementary material Fig.S1B).

The ability of SHP2 to regulate signaling and phenotypes ismodulated by elevated expression of EGFRvIIITo evaluate the regulatory functions of SHP2 in the context

of EGFRvIII expression, we stably depleted SHP2 in a panel ofU87MG cell lines that expressed low, medium or high levels ofEGFRvIII, or a high level of kinase-dead EGFRvIII (U87MG-L,

U87MG-M, U87MG-H or U87MG-DK, respectively) (Huanget al., 2007). SHP2 depletion reduced proliferation in all four celllines (Fig. 3A). Similar to effects observed in Fig. 1B, SHP2

knockdown also promoted survival in response to EGFR and c-MET co-inhibition in U87MG-DK, U87MG-L and U87MG-Mcells, but there was no effect in U87MG-H cells (Fig. 3B). To

confirm specificity of the effects of SHP2 knockdown, we used anadditional, non-overlapping SHP2 shRNA to deplete SHP2 inU87MG-M cells, where cells with SHP2 knockdown were againmore resistant to EGFR and c-MET co-inhibition (supplementary

material Fig. S2A). To understand the reason for the lack of effectof SHP2 knockdown on survival response in U87MG-H cells, wefirst investigated signaling pathways in resting cells by using

western blotting (Fig. 3C; supplementary material Fig. S2B).STAT3 phosphorylation was increased by SHP2 depletion in allcell lines including U87MG-H, which was surprising given our

previous findings that STAT3 controlled survival response and that

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SHP2 knockdown did not rescue U87MG-H cells followingtreatment with gefitinib and PHA665752. We also noticed reducedERK phosphorylation in all cell lines with SHP2 knockdown,

although the reduction in ERK phosphorylation in U87MG-H cellswas very modest – an effect which we explore further inexperiments shown in Fig. 4. In addition, we noticed potential

effects of SHP2 knockdown on the expression and phosphorylation

of EGFRvIII, and on phosphorylation of c-MET – findings whichwe also revisit later.

To delve further into the lack of effect of SHP2 knockdown on

cellular response to inhibitors in U87MG-H cells, we comparedthe effects of SHP2 knockdown in U87MG-M and U87MG-Hcells co-treated with gefitinib and PHA665752 (Fig. 3D;

supplementary material Fig. S2C). Interestingly, in the presence

Fig. 2. ERK and STAT3 pathways both control proliferation and survival of GBM cells. (A) U87MG, LN18, T98G and U118MG cells were seeded at100,000 cells/well and treated 24 h later with 6 mM CI-1040 (C), 4 mM Stattic (S) or DMSO as a control (DMSO) for 72 h prior to cell counting. Countsare represented as mean 6 s.e.m. (n53); *P,0.05. (B) The indicated cell lines were treated with the same concentrations of inhibitor as shown in A for 30 minprior to lysis. Lysates were analyzed by western blotting with antibodies against the indicated proteins. Images are representative of three sets of biologicalreplicates. (C) The indicated cell lines were treated with 6 mM CI-1040 (C) or 4 mM Stattic (S), as in A, or with 10 mM gefitinib + 1 mM PHA665752 (G+P), with10 mM gefitinib + 1 mM PHA665752 + 6 mM CI-1040 (G+P+C), with 10 mM gefitinib + 1 mM PHA665752 + 4 mM Stattic (G+P+S), or with DMSO as a control(DMSO). The gefitinib concentration in this set of experiments was lower than that used in the experiments shown in Fig. 1B and lower than the concentrationused in the experiments shown in panel D, in order to reduce cellular death in response to G+P treatment. After 96 h, the percentage of TO-PRO-3-positivecells was measured by flow cytometry (n53); *P,0.05. (D) The indicated cell lines expressing control or SHP2-targeting shRNA were treated with DMSO as acontrol (DMSO), with 4 mM Stattic (S), with 20 mM gefitinib + 1 mM PHA665752 (G+P), or with 20 mM gefitinib + 1 mM PHA665752 + 4 mM Stattic (G+P+S). After72 h, the percentage of TO-PRO-3-positive cells was measured by flow cytometry (n53); *P,0.05.

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of the inhibitors, SHP2 depletion significantly increased STAT3phosphorylation in U87MG-M cells but had essentially no effectin U87MG-H cells. Since STAT3 can be activated through direct

binding to EGFR (Shao et al., 2003; de la Iglesia et al., 2009)or EGFRvIII (Fan et al., 2013), we hypothesized that elevated

STAT3 Y705 phosphorylation is EGFRvIII-dependent inSHP2-depleted U87MG-H cells but EGFRvIII-independent inSHP2-depleted U87MG-M cells. This scenario would lead to

a preferential reduction in STAT3 phosphorylation with EGFRinhibition in U87MG-H cells. Consistent with this model,

Fig. 3. Sufficiently high EGFRvIII expression diminishes the ability of SHP2 to promote ERK activity and to reduce STAT3 phosphorylation in thepresence of EGFR and c-MET inhibitors. (A) U87MG-DK, U87MG-L, U87MG-M and U87MG-H cells expressing control or SHP2-targeting shRNA wereseeded at 75,000 cells/well and were counted 72 h later. Counts are represented as mean 6 s.e.m. (n53); *P,0.05. (B) The indicated cell lines expressingcontrol or SHP2-targeting shRNA were treated with DMSO as a control (DMSO) or co-treated with 20 mM gefitinib + 1 mM PHA665752 (G+P). After 72 h, thepercentage of TO-PRO-3-positive cells was measured by flow cytometry (n53); *P,0.05. (C) The indicated cell lines were grown in complete medium for 72 h,and lysates were analyzed by western blotting using antibodies against the indicated proteins. Images are representative of five sets of biologicalreplicates. (D) U87MG-M and U87MG-H cells expressing control or SHP2-targeting shRNA were co-treated with 20 mM gefitinib + 1 mM PHA665752 (G+P) for24 h and analyzed by western blotting using antibodies against the indicated proteins. Images are representative of three sets of biological replicates.(E) The indicated cell lines expressing control or SHP2-targeting shRNA were treated with DMSO as a control (DMSO) or with 20 mM gefitinib + 1 mMPHA665752 (G+P) for 24 h and lysed. STAT3 immunoprecipitates were analyzed by western blotting using antibodies against the indicated proteins. Images arerepresentative of three sets of biological replicates. (F) U87MG-M cells were analyzed as shown in panel B with the addition of 4 mM Stattic (S) where indicated(n53); *P,0.05.

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association of STAT3 and pEGFRvIII was much higher in U87MG-H cells than in any other line (Fig. 3E; supplementary material Fig.S2D). In U87MG-H cells, association of STAT3 and pEGFRvIII

was further promoted by SHP2 knockdown, presumably because ofthe concomitant increase in phosphorylated levels of EGFRvIIIobserved in this cell line. The unchanged association of STAT3 and

pEGFRvIII for SHP2-depleted U87MG-M cells is consistent withthe notion that EGFRvIII is not a primary driver of STAT3phosphorylation below a threshold level of EGFRvIII expression.

Moreover, co-treatment with gefitinib and PHA665752 eliminatedassociation of STAT3 and pEGFRvIII in U87MG-H cells,

consistent with reduced STAT3 phosphorylation in response togefitinib and PHA665752 co-treatment (Fig. 3E). It should also benoted that EGFRvIII and c-MET phosphorylation were greatly

reduced in cells co-treated with gefitinib and PHA665752 relative toDMSO-treated cells with or without SHP2 knockdown, whicheliminates the possibility that a failure to reduce receptor

phosphorylation was responsible for the resistance to EGFR andc-MET co-inhibition in cells with SHP2 knockdown(supplementary material Fig. S2E). Finally, as in parental

U87MG cells, combining Stattic with gefitinib and PHA665752enhanced cell death in SHP2-depleted U87MG-M cells (Fig. 3F).

Fig. 4. Sufficiently high EGFRvIII expression suppresses EGF-mediated ERK phosphorylation by SHP2 sequestration. (A) Serum-starved U87MG-DK,U87MG-L, U87MG-M and U87MG-H cells were treated with or without 10 ng/ml EGF for 5 min and lysed. SHP2 immunoprecipitates were analyzed bywestern blotting using antibodies against the indicated proteins. Images are representative of three sets of biological replicates. (B) Membrane and cytosolicfractions from serum-starved U87MG-DK and U87MG-H cells were analyzed by western blotting using antibodies against indicated proteins. Images arerepresentative of four sets of biological replicates. (C) Serum-starved U87MG-L and U87MG-H cells were treated with or without 10 ng/ml EGF for 5 min, andslides were prepared for SHP2 immunofluorescence. Images are representative of multiple frames from three biological replicates. (D) EGF endocytosisrate constants (ke) were measured for the indicated cell lines by using 125I-EGF. Data are represented as mean 6 s.e.m. (n56); *P,0.05. (E) The indicatedserum-starved cell lines were treated with 10 ng/ml EGF for up to 15 min and lysates were analyzed by western blotting using antibodies against the indicatedproteins. Images are representative of three sets of biological replicates.

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At high expression levels EGFRvIII is able to sequester SHP2In the same way that STAT3 can preferentially bind to EGFRvIII,

other proteins may be sequestered by EGFRvIII when thereceptor is expressed at high levels. We hypothesized that such apreferential binding effect for SHP2 could explain the modesteffect that knockdown of SHP2 has on ERK phosphorylation in

U87MG-H cells, as well as the gradual reduction in basalphosphorylation of ERK in control cells with increasingexpression of EGFRvIII (Fig. 3C). Such an effect would be

analogous to one we described in lung cancer cells, wherein kinase-activated and internalization-impaired EGFR mutants appear tosequester adapter-bound SHP2 in such a way that full ERK

activation is prevented (Furcht et al., 2012). To explore this, wefirst investigated the basal and EGF-induced associations of SHP2with GAB1 and with EGFRvIII. Increasing EGFRvIII expression

clearly promoted association of GAB1 and SHP2 (Fig. 4A;supplementary material Fig. S3A), as well as the previouslyreported association of pEGFRvIII and SHP2 (Zhan et al., 2009).Although association of GAB1 and SHP2 was EGF-inducible in

U87MG-DK, U87MG-L and U87MG-M cells, the high basalassociation observed in U87MG-H cells was so elevated that it wasnot augmented further by treatment with EGF. We further noted a

larger fraction of SHP2 in the membrane compartment along withEGFRvIII in U87MG-H cells than in U87MG-DK cells (Fig. 4B;supplementary material Fig. S3B); an ,1.5-fold increase of the

SHP2 signal within the membrane compartment was observed inU87MG-H cells relative to U87MG-DK cells. Thus, the activity ofEGFRvIII can promote sequestration of SHP2 in the membrane

fraction. We also found that elevated EGFRvIII expression alteredthe basal intracellular distribution of SHP2, as observed byimmunofluorescence. Whereas SHP2 moved from the cell interiortowards the cell periphery in response to EGF in U87MG-L cells,

basal SHP2 was already peripherally distributed in U87MG-H cellsand did not redistribute in response to EGF (Fig. 4C;supplementary material Fig. S3C,D). Given that EGF-mediated

endocytosis of wild-type EGFR was significantly reduced inU87MG-H cells (Fig. 4D) and that EGFRvIII itself is alsoendocytosis impaired (Huang et al., 1997; Grandal et al., 2007),

our data are, indeed, consistent with the notion that active adapter-and EGFRvIII-bound SHP2 is sequestered at the plasma membranein U87MG-H cells. The effect of this on ERK activation seems sopronounced that ERK phosphorylation cannot be induced in

U87MG-H cells by exogenous EGF, whereas ERK inductiondoes occur in U87MG-DK, U87MG-L and U87MG-M cells(Fig. 4E; supplementary material Fig. S3E). To explore whether

altered expression of MKP3, the primary phosphatase for ERK, isresponsible for the failure of EGF to induce ERK phosphorylationin U87MG-H cells, we investigated MKP3 expression across the

panel of cell lines. However, we observed no trends in MKP3expression that would explain our data (supplementary materialFig. S3F).

SHP2 negatively regulates phosphorylation of EGFRvIII andc-METAs previously noted, the results shown in Fig. 3C suggest the ability

for SHP2 to regulate EGFRvIII and c-MET phosphorylation.Specifically, the data show that total levels of phosphorylatedEGFRvIII and c-MET were increased when SHP2 was knocked

down in U87MG-H cells, but that EGFRvIII phosphorylation wasreduced when SHP2 was knocked down in U87MG-L andU87MG-M cells. The apparent effect in U87MG-L and U87MG-

M cells might arise because of the concomitant decrease in

EGFRvIII expression when SHP2 is knocked down, which mightresult from impaired ERK activity (supplementary material Fig.

S4A). To clarify this further, we ectopically expressedconstitutively active SHP2 (SHP2E76A) in all four cell lines. Thishad a minimal effect on EGFRvIII expression but increased ERKphosphorylation and reduced EGFRvIII, c-MET and STAT3

phosphorylation (Fig. 5A; supplementary material Fig. S4B).SHP2E76A expression also promoted cell sensitivity to gefitiniband PHA665752 co-treatment in U87MG-M cells (Fig. 5B). To

further investigate how SHP2 regulates EGFRvIII, c-MET andSTAT3 phosphorylation, we transiently expressed SHP2WT or thesubstrate-trapping SHP2 double mutant SHP2D425A/C459S

[SHP2DM; (Agazie and Hayman, 2003)] in all four U87MG celllines. This double mutation abrogates the catalytic activity of SHP2and causes irreversible binding of the catalytic domain to substrates.

Phosphorylated EGFRvIII and c-MET co-immunoprecipitated withSHP2DM (Fig. 5C; supplementary material Fig. S4C), but STAT3did not (supplementary material Fig. S4D), suggesting thatEGFRvIII and c-MET are substrates of SHP2. These specific

interactions have not been reported previously, but it has beenreported that SHP2 can directly dephosphorylate other receptors,including HER2 (Zhou and Agazie, 2009), based on experiments

using SHP2DM.

SHP2 knockdown impedes tumor xenograft growth andexpression of hypoxia-inducible factorsFemale Nu/Nu immunodeficient nude mice were injectedsubcutaneously in both flanks with U87MG-M control or SHP2

knockdown cells. Tumors arising from control cells grew well andwere highly responsive to gefitinib and PHA665752 co-treatment(Fig. 6A,B), suggesting that this co-treatment strategy can beeffective in EGFRvIII-expressing GBM. We had hoped to be able

to grow tumors arising from SHP2 knockdown cells to investigatethe potential ability of SHP2 depletion to promote tumor resistanceto gefitinib and PHA665752 co-treatment. However, after reaching

an average maximum volume of 40 mm3, tumors arising from cellswith SHP2 knockdown gradually shrank and never reached asufficient size to begin treatment (Fig. 6B). Interestingly, HIF-1/2aexpression was reduced in tumors arising from SHP2 knockdowncells compared with controls (Fig. 6C), which may explain theirfailure to form tumors. In vitro studies revealed a similar effect ofSHP2 knockdown on HIF-2a expression under hypoxic and

normoxic conditions, and on HIF-1a expression for normoxicculture (Fig. 6D). Control cells treated with the MEK inhibitorU0126 displayed diminished HIF-1a expression in normoxia and

HIF-2a expression in both normoxia and hypoxia, suggesting thatSHP2 regulates HIF-1/2a expression through controlling ERKactivity.

DISCUSSIONOur results demonstrate for the first time that the ability of SHP2

to exert multivariate control over signaling in GBM cells enablesit to regulate simultaneously and differentially the phenotypes ofproliferation and resistance to therapy. This effect arises, at leastin part, because ERK and STAT3, which are regulated in

qualitatively different ways by SHP2, play dominant roles inthe regulation of proliferation and therapeutic resistance,respectively. We uncovered a number of other previously

undocumented SHP2 regulatory functions, including SHP2-mediated antagonism of EGFRvIII and c-MET phosphorylation,and regulation of HIF-1/2a expression, which may also play roles

in determining GBM cell and tumor phenotypes. These integrated

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SHP2 signaling mechanisms and the ways they impact GBM cellphenotypes are summarized in Fig. 7.

A focus of our study is the impact of the ability of SHP2 tosimultaneously promote ERK activity and suppress STAT3

phosphorylation. It has been shown in other contexts, and weshow it explicitly for GBM cells, that the ERK and STAT3pathways both promote proliferation and survival (here in

response to co-inhibition of EGFR and c-MET). Overlap in thecontrol of transcriptional events by ERK and STAT3 helps toexplain their overlapping control of phenotypes. For example,

ERK and STAT3 both drive expression of proteins that promotecell cycle progression and proliferation, promote expression ofanti-apoptotic proteins and downregulate proteins in apoptotic

pathways (Zhang and Liu, 2002; Yu and Jove, 2004; Dauer et al.,2005; Lu and Xu, 2006; Krens et al., 2008). This functionaloverlap in the regulation of broad classes of genes also containsoverlap of specific gene products such as VEGF (Yu and Jove,

2004; Krens et al., 2008) and c-MYC (Zhang and Liu, 2002; Yu

and Jove, 2004). Even with this partial overlap, in GBM cell linesthe positive regulation of ERK by SHP2 is dominant indetermining the effect of SHP2 expression on cellularproliferation, whereas SHP2-mediated suppression of STAT3

dominates in determining the effect of SHP2 expression oncellular sensitivity to co-inhibition of EGFR and c-MET. Thisupdated view of the consequences of the multivariate control of

SHP2 regarding signaling and phenotype fits within the generalparadigm that cells integrate and interpret multivariate signalinginformation in different ways in the execution of cellular

decisions (Janes et al., 2005; Kumar et al., 2007; Lazzara andLauffenburger, 2009).

Other new aspects of our findings include the discovery that

sufficiently high expression of EGFRvIII reduces the antagonismof STAT3 phosphorylation in the presence of kinase inhibitors, inaddition to reducing the contribution of SHP2 to ERK activation,and the mechanisms underlying these effects. Others have noted a

suppression of ERK activity when EGFRvIII is expressed

Fig. 5. Expression of SHP2 mutants reveals negative regulation of EGFRvIII, c-MET and STAT3 phosphorylation. (A) Lysates of U87MG-DK, U87MG-L,U87MG-M and U87MG-H cells transduced with an empty pBabe.Puro vector (pBP) or SHP2E76A (E76A) and grown in full medium for 72 h were analyzedby western blotting using antibodies against the indicated proteins. Images are representative of three sets of biological replicates. (B) U87MG-M cellstransduced with pBP or SHP2E76A were treated with DMSO as a control (DMSO) or with 20 mM gefitinib + 1 mM PHA665752 (G+P). After 72 h, the percentage ofTO-PRO-3-positive cells was measured by flow cytometry (n53); *P,0.05. (C) Serum-starved cells of the indicated cell lines transiently transfected withSHP2WTor the double mutant SHP2D425A/C459S (SHP2DM) were lysed. SHP2 immunoprecipitates were analyzed by western blotting using antibodies against theindicated proteins. Images are representative of three sets of biological replicates.

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(Moscatello et al., 1996), but the mechanistic basis for this hadnot previously been explored. Our data suggest that this effect isrelated to a mechanism we have elucidated previously for

structurally distinct EGFR mutants in NSCLC cells, wherein thekinase-activated EGFR mutants, which display reduced ability toactivate ERK and a reduced rate of ligand-mediated endocytosis,

also promote basal sequestration of SHP2 with EGFR and GAB1at the cell periphery (Lazzara et al., 2010; Furcht et al., 2012). Itis also interesting to note that in NSCLC cells that express wild-type EGFR, SHP2 knockdown promotes an enhanced response to

gefitinib (Furcht et al., 2012) rather than resistance. Thispresumably occurs because SHP2 knockdown producesgenerally small effects on STAT3 Y705 phosphorylation in

NSCLC cells (Furcht et al., 2012) compared with what weobserved here in GBM cell lines, further highlighting the

contextual dependence of the functions of SHP2. Interestingly,the ability of EGFRvIII to sequester proteins also underlies lossof SHP2 control of STAT3 phosphorylation in the presence of

EGFR and c-MET inhibitors at high EGFRvIII levels. As wedemonstrate in Fig. 3E, this effect involves a shift in the ability ofEGFRvIII to bind STAT3 when EGFRvIII is expressed at the

highest levels. It is also worth noting that the ability of SHP2 tonegatively regulate EGFR and c-MET phosphorylation was mostapparent when EGFRvIII was expressed at high levels. This couldalso result from sequestration of active SHP2 at the plasma

membrane where it has ready access to these receptors. We alsonote that the range of EGFRvIII expression explored here isconsistent with that observed in tumors (Moscatello et al., 1995).

Thus, the dependence of SHP2 functions on EGFRvIII expressionmight be clinically relevant.

Fig. 6. Gefitinib and PHA665752 co-treatment or SHP2 knockdown impairs U87MG tumor xenograft growth. Mice were subcutaneously injected withcontrol or SHP2-depleted U87MG-M cells. When tumors reached an average size of 50 mm3 (control shRNA only), mice were treated daily either withvehicle or 100 mg/kg gefitinib + 30 mg/kg PHA665752 (G+P) for 7 days (black arrow indicates the start of the treatment). (A) After treatment concluded, pictureswere taken and tumors were harvested. (B) Tumor volumes were measured before and throughout treatment. Data are shown as mean 6 s.e.m. (controlshRNA: vehicle, SHP2 shRNA: no treatment and control shRNA: G + P; n512, 26 and 14 tumors, respectively). (C) Tumor lysates were analyzed by westernblotting using antibodies against the indicated proteins. Densitometry data are shown as mean 6 s.e.m. (n53). (D) U87MG-M cells expressing control or SHP2-targeting shRNA were pretreated with DMSO or 40 mM U0126 (control shRNA only) for 24 h prior to hypoxic culture for 24 h. Lysates were analyzed by westernblotting using antibodies against the indicated proteins. Densitometry data are shown as mean 6 s.e.m. (n53); *P,0.05.

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There are, of course, additional signaling pathways regulatedby EGFRvIII and SHP2 that have not been explored here but

which could play roles in some apparent quantitativeinconsistencies between the effects of altered SHP2 expressionon signaling pathways and the phenotypic roles we have ascribed tothose pathways. One example pertains to our observations in Fig. 3

in U87MG-H cells, where control cells were highly proliferativedespite displaying relatively low basal phosphorylation of ERK,and SHP2 knockdown produced a modest effect on ERK

phosphorylation but a large effect on proliferation. As just onepossible explanation for this, the increased abundance of EGFRvIIIin U87MG-H cells might promote the activity of other pathways

that could compensate for ERK in promoting proliferation. If theactivities of those other pathways are also regulated by SHP2 in away that promotes proliferation, a large drop in proliferation could

still accompany SHP2 knockdown with only a modest effect onERK. In the future, it will be important to investigate these issuesmore broadly and quantitatively to fully understand the potentialimpact of targeting SHP2 or EGFRvIII in GBM.

Our data on the effects of SHP2 knockdown, expression ofconstitutively active SHP2 and expression of substrate-trappingSHP2 all suggest that SHP2 negatively regulates EGFRvIII

phosphorylation, potentially through direct interaction. Thiscontrasts with previous reports that SHP2-mediated ERKactivity increases levels of phosphorylated EGFRvIII (Zhan and

O’Rourke, 2004). This discrepancy might be explained by ouradditional finding that SHP2-mediated ERK activity enhancesEGFRvIII expression, an effect that has also been noticed by

others when knocking down SHP2 in certain GBM cell lines(Zhan et al., 2009). Thus, SHP2 appears to exert twocountervailing effects, either of which might be dominant, inthe determination of total cellular phosphorylated EGFRvIII

levels. The notion that SHP2 can negatively regulate EGFRvIIIphosphorylation might seem at odds with our finding that SHP2

knockdown impairs xenograft growth or the analogous findingsof Zhan et al. (Zhan et al., 2009) where a catalytically inactive

SHP2 was expressed in an EGFRvIII-positive tumor model. Weinterpret these aggregate results as indicating that any potentialability for SHP2 to impair tumorigenesis by negatively regulatingEGFRvIII phosphorylation is trumped by the positive regulatory

functions of SHP2 in tumorigenesis, including its apparent abilityto control HIF-1/2a expression, at least in the cell line model usedhere.

Given ongoing efforts to develop specific SHP2 inhibitors forclinical use, it is worth noting that two distinct effects of SHP2inhibition could arise in GBM cells and tumors. Based on our

results, SHP2 inhibition would be expected to inhibit ERKactivity but, simultaneously, to promote STAT3 phosphorylation.In cell culture, the integrated effect of these signaling

perturbations was to slow cell growth while simultaneouslypromoting resistance to EGFR and c-MET co-inhibition. Basedon this alone, it is unclear whether SHP2 inhibition is a usefultherapeutic approach. Our finding that SHP2 controls expression

of HIF-1/2a and growth of GBM tumor xenografts may obviatepotential concerns about the ability of SHP2 inhibition to promotesurvival signaling through the STAT3 pathway; however, this

remains to be demonstrated in more-detailed GBM tumor modelsthat explore the potential ability of very high EGFRvIIIexpression to modulate SHP2 function. Assuming that SHP2

function is, indeed, controlled by EGFRvIII levels in vivo, recentadvances in detecting EGFRvIII protein through magneticresonance imaging (Tykocinski et al., 2012), as opposed to

traditional tumor tissue biopsy approaches, may eventuallyadvance our ability to predict the impact of SHP2 inhibition.

It should also be noted that our xenograft studies show promisefor combining EGFR and c-MET inhibitors in EGFRvIII-positive

GBM. This has not been previously demonstrated in vivo, althougha previous study demonstrated the suitability of combining anHGF-targeted antibody with an EGFR inhibitor (Lal et al., 2009).

Interestingly, the usefulness of certain irreversible EGFR inhibitorsin EGFRvIII-positive GBM may obviate the need to combine c-MET and EGFR inhibitors (Vivanco et al., 2012). Whether or not

c-MET inhibitors are needed moving forward, SHP2 inhibitorsmight eventually offer an attractive alternative in the treatment ofGBM where resistance to other inhibitors arises. Of course, ourdata support the potential usefulness of STAT3 inhibitors in

treating GBM. STAT3 has previously been identified as a keyregulator of GBM cell survival (Lo et al., 2008; de la Iglesia et al.,2009) and at least one clinical trial (ClinicalTrials.gov

NCT01904123) is scheduled to begin recruiting patients later thisyear to test the efficacy of STAT3 inhibition in cancers includingGBM.

MATERIALS AND METHODSCell cultureLN18, T98G and U118MG cells were obtained from the American Type

Culture Collection (Manassas, VA). U87MG parental cells and cells

expressing low (16106 receptors/cell), medium (1.56106 receptors/cell)

or high (26106 receptors/cell) levels of EGFRvIII (U87MG-L, U87MG-

M or U87MG-H, respectively), or a dead-kinase mutant of EGFRvIII

(U87MG-DK, 26106 receptors/cell) were a generous gift from Frank

Furnari (University of California San Diego, San Diego, CA). All cells

were maintained in DMEM supplemented with 10% FBS, 1 mM L-

glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. U87MG

cells expressing EGFRvIII were cultured with 400 mg/ml G418. Where

indicated, cells were treated with EGF (Peprotech, Rocky Hill, NJ)

following 8–16 h starvation in medium containing 0.1% FBS. All cell

Fig. 7. Summary of the oncogenic and anti-survival functions of SHP2in GBM cells. Consistent with its best-described role, SHP2 possessesoncogenic functions by promoting the phosphorylation of ERK, whichaugments expression of EGFRvIII and HIF-1/2a. Conversely, SHP2antagonizes survival signaling by apparent activity against EGFRvIII andc-MET as well as negative regulation of multiple modes of STAT3phosphorylation.

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culture reagents were from Life Technologies (Carlsbad, CA). For

experiments under hypoxic conditions, cells were cultured for 24 h in

0.5% O2 by using an Invivo2 400 hypoxia workstation (Ruskinn

Technology, Grandview, MO) prior to lysis.

shRNA and stable expression constructsDNA oligonucleotides encoding short hairpins targeting human SHP2

(Integrated DNA Technologies, San Jose, CA) were cloned into the

pSicoR vector (Tyler Jacks, MIT, Cambridge, MA) (Ventura et al.,

2004). The main shRNA targeted nucleotides 1780–1798 of SHP2

mRNA (59-GGACGTTCATTGTGATTGA-39). Control vectors were

created by using shRNA sequences that do not target a known human

mRNA. We also used an alternative, non-overlapping SHP2 shRNA

targeting nucleotides 5890–5908 (59-GTATTGTACCAGAGTATTA-39)

of human SHP2 in a cell proliferation experiment in the presence of drugs

(supplementary material Fig. S2A). Combined with the data showing the

effects of SHP2E76A expression, the data in supplementary material Fig.

S2A help to demonstrate the SHP2 specificity of effects observed using

the primary SHP2-targeting hairpin. To engineer cells that express

shRNA, lentivirus was produced by using calcium-phosphate-mediated

transfection of 293FT cells (Life Technologies) with pSicoR plasmids

together with the packaging plasmids pCMV-VSV-G, pMDL-gp-

RRE, and pRSV-Rev (Marilyn Farquhar, University of California San

Diego, San Diego, CA). Virus was collected 48 and 72 h post-

transfection and filtered using 0.45 mm syringe filters prior to infecting

target cells.

SHP2 cDNA encoding SHP2E76A (Ben Neel, Ontario Cancer Institute,

Toronto, ON, Canada) was inserted at the EcoRI site of the pBabe vector.

Retrovirus was produced by calcium phosphate-mediated transfection of

amphotropic Phoenix cells (Gary Nolan, Stanford University, Stanford,

CA) with vector. Virus was harvested 24, 30 and 48 h post-transfection

and used to infect target cells, which were selected in 2 mg/ml puromycin

(Sigma-Aldrich, St Louis, MO). All expression and shRNA constructs

were validated by sequencing, and protein knockdown was validated by

western blot.

Transient expression of wild-type or substrate-trapping SHP2Expression constructs for wild-type and substrate-trapping double mutant

(DM; D425A/C459S) SHP2 in the pMT2 vector backbone were provided

by Yehenew Agazie (West Virginia University, Morgantown, WV).

U87MG cells were transfected with pMT2 plasmids using calcium

phosphate. Cells were lysed in immunoprecipitation lysis buffer (Cell

Signaling Technology, Danvers, MA; #9803) 48 h after transfection.

InhibitorsStocks of gefitinib (LC Laboratories, Woburn, MA), U0126 (LC

Laboratories), CI-1040 (LC Laboratories), PHA665752 (Santa Cruz

Biotechnologies, Dallas, TX) and Stattic (Sigma-Aldrich) were prepared

in DMSO.

Cell death quantificationCells were seeded at a density of 75,000–100,000 cells/well in six-well

dishes, and treated 24 h later with different combinations of gefitinib,

PHA665752, CI-1040 and Stattic, or with DMSO (control). After 72–

96 h, floating and adherent cells were pooled and stained for permeability

to TO-PRO-3 (Life Technologies). Cells were analyzed by flow

cytometry within 1 h of resuspension. Flow cytometry was performed

on a Becton Dickinson Biosciences FACSCalibur cytometer and data

were analyzed using FlowJo.

Proliferation measurementsCells were seeded at an initial density of 75,000 or 100,000 cells/well in

six-well dishes. After growing for 72 h, cells were trypsinized, suspended

in complete medium and counted by using a hemocytometer.

XTT viability assayCell proliferation in the presence of inhibitors was assessed using the

XTT Cell Proliferation Assay (Roche, Indianapolis, IN). Cells were

seeded in 96-well plates, grown for 16–24 h, and switched to complete

medium containing up to 20 mM gefitinib and/or 1 mM PHA665752 for

an additional 3 days. Subsequently, fresh medium and XTT reagent were

added to wells, and plates were incubated for 2–4 h at 37 C to maximize

signal-to-background. Absorbance was measured at 450 nm with a

reference wavelength at 660 nm. The percentage of viable cells was

determined by normalizing absorbance to that of cells treated with

DMSO. Each experiment was performed on at least three separate days

with each condition plated in three replicate wells on each day, except

where noted.

Tumor xenograftsFemale Nu/Nu immunodeficient nude mice (Charles River, Wilmington,

MA) were subcutaneously injected in both flanks with control or SHP2-

depleted U87MG-M cells (control shRNA: 750,000 cells; SHP2 shRNA:

2,500,000 cells). The difference in injected cell numbers was based upon

observations of different rates of proliferation in vivo and in vitro with or

without SHP2 knockdown. When tumors reached an average size of

50 mm3 (achieved by control tumors only), 7 day treatment with

gefitinib and PHA665752 (Selleck Chemicals, Houston, TX) began.

Gefitinib was resuspended in an aqueous solution containing 0.5%

hydroxypropylmethylcellulose (Sigma-Aldrich) and 0.1% Tween-80

(Sigma-Aldrich) and was delivered at 100 mg/kg/day daily by oral

gavage. PHA665752 was resuspended in an aqueous solution containing

1% dimethyl acetamide (Sigma-Aldrich), 10% propylene glycol (Sigma-

Aldrich), and 1.05 moles L-lactic acid (Sigma-Aldrich) per mole of

PHA665752, and was delivered at 30 mg/kg/day daily by intraperitoneal

injection. Tumors were measured with a caliper before and throughout

treatment, and tumor volume was calculated as p/66A6B2, where A and

B are the larger and smaller tumor diameters, respectively. Excised

tumors were homogenized in immunoprecipitation lysis buffer before

proceeding with western blotting. All experiments were approved by the

University of Pennsylvania Institutional Animal Care and Use

Committee and performed in accordance with NIH guidelines.

Subcellular fractionationSerum-starved cells were washed with PBS and collected in hypotonic

buffer (10 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM EDTA)

supplemented with 1 mM PMSF, additional protease inhibitors, and

phosphatase inhibitors. Crude lysates were generated with a Dounce

homogenizer and centrifuged at 800 and 7900 g, for 5 min at each speed,

to remove nuclei and mitochondria, respectively. Cleared lysates were

centrifuged at 100,000 g for 60 min to separate membrane and cytosol

fractions. Membrane pellets were washed with PBS, resuspended in

hypotonic buffer and centrifuged again at 100,000 g. After additional

washes, membrane pellets were resuspended in immunoprecipitation

lysis buffer to solubilize proteins. To improve signals, membrane

fractions were 106 more concentrated than cytosolic fractions in terms

of the relative amount of total lysate loaded.

EGF internalization assayEGF-mediated EGFR endocytosis rate constants (ke) were measured

using 125I-EGF as described previously (Wiley and Cunningham, 1982;

Lund et al., 1990).

Western blottingWhole-cell lysates were prepared in a standard cell extraction buffer

(Life Technologies; FNN0011) prepared with protease inhibitors

and phosphatase inhibitors (Sigma-Aldrich). Lysates were cleared

by centrifugation at 16,100 g for 10 min, and total protein

concentrations were determined by micro-bicinchoninic acid (BCA)

assay (Thermo Fisher Scientific, Waltham, MA). Proteins were resolved

on 4–12% gradient polyacrylamide gels (Life Technologies) under

denaturing and reducing conditions and transferred to 0.2 mm

nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA).

Membranes were imaged using a LI-COR Odyssey Imaging System.

When needed, membranes were stripped with 0.2 M NaOH.

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ImmunofluorescenceCells were seeded at 150,000 cells/well on 18 mm glass coverslips in six-

well culture dishes. After serum starvation for 16 h, cells were treated

with 10 ng/ml EGF for up to 30 min. Cells were then washed and fixed

for 20 min in 4% paraformaldehyde and permeabilized with 0.25%

Triton X-100 in PBS for 5 min. Coverslips were again washed and

incubated with the SHP2 antibody in a humidified chamber for 3 h

at 37 C. Washed coverslips were incubated with Alexa-Fluor-488-

conjugated secondary antibody (Life Technologies) and Hoechst DNA

stain (Sigma-Aldrich) for 1 h at 37 C. Coverslips were mounted on

microscope slides using Prolong Gold Antifade mounting media (Life

Technologies) and dried overnight. Fixed slides were imaged on a Zeiss

Axiovert 40 CFL microscope using an A-Plan 1006 oil objective and a

SPOT Insight CCD camera. Specificity of the SHP2 antibody for

immunofluorescence was verified using U87MG-M cells with or without

SHP2 knockdown (supplementary material Fig. S3C).

ImmunoprecipitationCells were lysed in immunoprecipitation lysis buffer, with 1 mM PMSF,

protease inhibitors, and phosphatase inhibitors. After lysate centrifugation

at 16,100 g and 4 C for 10 min and determination of protein concentrations

by BCA assay, 400–500 mg of protein was incubated with agarose beads

conjugated to SHP2 or STAT3 antibodies at 4 C overnight. Beads were

washed three times with cold lysis buffer, re-suspended in LDS sample

buffer (Life Technologies) and boiled before western blotting.

Immunoprecipitation specificity was validated with comparisons to a

rabbit control antibody (IgG; Santa Cruz Biotechnology) (supplementary

material Figs S2D, S3A, S4C).

AntibodiesAntibodies against EGFR (catalog no. 2232), c-MET pY1234/1235

(catalog no. 3126), STAT3 pY705 (catalog no. 9145), ERK (catalog no.

4695) and ERK pT202/Y204 (catalog no. 4377) were from Cell Signaling

Technology. SHP2 (sc-280) and STAT3 (sc-482) antibodies were from

Santa Cruz Biotechnology. Antibodies against actin (MAB1501) and

GAB1 (catalog no. 06-579) were from Millipore (Billerica, MA).

Antibody against EGFR pY1068 (catalog no. 1727) was from Epitomics

(Burlingame, CA). Antibodies against HIF-1a (catalog no. 10006421)

and HIF-2a (NB100-122) were from Cayman Chemical (Ann Arbor,

MI) and Novus Biologicals (Littleton, CO), respectively. Infrared dye-

conjugated secondary antibodies were from Rockland Immunochemicals

(Gilbertsville, PA). All antibodies were used according to manufacturers’

recommendations.

StatisticsStatistical analyses were performed using a paired two-tailed Student’s

t-test.

AcknowledgementsWe thank Donald M. O’Rourke for reviewing the manuscript and providing helpfulfeedback. We thank Ben Neel, Tyler Jacks, Marilyn Farquhar, Gary Nolan, FrankFurnari, and Yehenew Agazie for generously providing reagents.

Competing interestsThe authors declare no competing interests.

Author contributionsC.M.F., J.M.B. and A.M.R. performed cell culture experiments. N.S., L.K.M.,C.M.F. and J.M.B. performed mouse studies. C.M.F., J.M.B., M.C.S. and M.J.L.designed experiments. C.M.F., J.M.B. and M.J.L. wrote the manuscript.

FundingC.M.F. was supported in part by the University of Pennsylvania Cell and MolecularBiology Training Grant [grant number T32 GM-07229]; Training Program inCancer Pharmacology [grant number R25 CA101871-07]; and a fellowship fromthe Ashton Foundation. J.M.B. was supported in part by the National Institutes ofHealth under Ruth L. Kirschstein National Research Service Award2T32HL007954 from the NIH-NHLBI; and the National Science FoundationGraduate Research Fellowship [grant number DGE-08220]. A.M.R. received

support from the University of Pennsylvania Institute for Regenerative Medicine.This project was funded, in part, under a grant with the Pennsylvania Departmentof Health. The Department specifically disclaims responsibility for any analyses,interpretations or conclusions. This work was also supported in part by funds fromthe Howard Hughes Medical Institute and funds to M.J.L. from the University ofPennsylvania. Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.150862/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2014) 127, 3555–3567 doi:10.1242/jcs.150862

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