MET amplification as a mechanism of acquired resistance to EGF receptor (EGFR)-targeted therapies in non–small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by coexpression of ERBB3. EGFR–MET cross-talk was not direct, but occurred by a combined regulation of MET levels and intermediary signaling through mitogen-activated protein kinases (MAPK). In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAPK reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Finally, EGFR–MET signaling was enhanced in a highly metastatic EGFR-mutant cell subpopulation, compared with the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR–MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression. Cancer Res; 73(16); 5053–65. ©2013 AACR.

Activating mutations in EGF receptor (EGFR) are commonly found in non–small cell lung carcinoma (NSCLC), and cells expressing mutant alleles depend on EGFR signaling for survival. Drugs targeting the EGFR kinase domain are effective for NSCLCs; unfortunately, responding tumors eventually progress owing to acquired resistance. Mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKI) include secondary mutations in EGFR and activation of compensatory receptor tyrosine kinases (RTK), such as MET. In 5% to 20% of recurrent patients, MET activation sustains tumor cell survival and is associated with relapse (1–4). Accordingly, EGFR and MET combination therapies are in clinical trials for patients harboring MET amplification and resistance to EGFR TKIs (5).

Bidirectional signaling between EGFR and MET occurs in both EGFR TKI-resistant and drug-naïve NSCLC cells. Notably, in NSCLC cells addicted to EGFR mutations, EGFR inhibition reduces basal MET phosphorylation (6–8). Conversely, MET inhibition in cells with high basal MET activity or MET amplification reduces basal phosphorylation of EGFR and ERBB family members, ERBB2 and ERBB3 (1, 2, 9, 10). In MET-amplified cells, MET signaling through ERBB3 maintains PI3K/Akt cell survival signaling despite EGFR inhibition (1). In addition, in NSCLC with wild-type EGFR or EGFR TKI resistance mutations, ligand stimulation of EGFR induces MET activation, and vice versa (8, 11, 12).

Mechanisms and outcomes of these biochemical events are difficult to interpret owing to the complexity of inter-ERBB interactions, which may involve promiscuous ERBB heterodimerization or indirect signaling cascades. Proposed mechanisms of ERBB–MET cross-talk include direct interaction, activation through autocrine regulation, and signaling through intermediary proteins (12, 13). Moreover, receptor cross-talk in some cells may not be bidirectional, but rather unilateral from EGFR to MET (14). In cancer cells encoding wild-type EGFR, MET regulates invasion and cell motility in an EGF-dependent manner (8, 12). Significantly, it is unknown if EGFR–MET cross-talk in lung cancers with EGFR mutations modulates similar phenotypes, particularly in patients with advanced disease and metastatic relapse. Defining the specific biologic contexts of ERBB–MET cross-talk, identifying their mechanisms of action, and characterizing their function may help expand clinical settings where combinatorial EGFR-MET therapies would benefit patients with NSCLC.

To systematically examine ERBB–MET cross-talk, we used a cellular system enabling analysis of functional interactions between individual ERBBs and MET in isolation or combination. We found that EGFR is sufficient to stabilize and cross-activate MET in the absence of other ERBBs. Activation is indirect, involving MEK/p38MAPK signaling, and is enhanced by ERBB3. This EGFR–MET axis is also active in NSCLC cells, where MET facilitates EGF-induced migration and invasion irrespective of EGFR mutational status. Finally, EGFR–MET activation is enhanced in an experimental model of metastatic NSCLC cells and potentiates brain metastasis.

Cell culture

32D mouse myeloid cells [American Type Culture Collection (ATCC)] were grown in RPMI with 10% heat-inactivated FBS and 10% WEHI-3B conditioned medium (CM). WEHI-3B (ATCC) cells were grown in RPMI with 10% heat-inactivated FBS. For WEHI-CM, WEHI-3B cells were grown in RPMI with 1% FBS for 4 days, medium was collected, and passed through a 0.22 μm filter. A549, H441, H2030, H1650, HCC827, H1975 cells (ATCC), PC9, and PC9-BrM3 cells (15) were grown in RPMI with 10% FBS. SYF−/− mouse embryonic fibroblasts (MEF; ref. 16) were grown in Dulbecco's Modified Eagle Medium with 10% FBS. All media contained 1% penicillin/streptomycin, l-glutamine, and sodium pyruvate.

Cloning

Site-directed mutagenesis was conducted with QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer's protocol. Primers were designed using the manufacturer's guidelines and obtained from Integrated DNA Technologies. The pRK5TKneo-MET-V5/His (Genentech) plasmid was used to make pRK5TKneo-MET-K1110A-V5/His, pRK5TKneo-MET-Y1234/1235F-V5/His, and pRK5TKneo-MET-Y1349/1354F-V5/His.

Plasmids, electroporation, and virus production

32D cells were electroporated at 240 V, 1 pulse, 35 milliseconds, with pBABE-EGFR (Addgene), pBABE-ERBB3 (Nancy Hynes, Friedrich-Miescher Institute, Basel, Switzerland), pBABE-EGFR-L858R (Don Nguyen, Yale University School of Medicine, New Haven, CT), pCDNA3(-)-EGFR-L858R/T790M (Katerina Politi, Yale University, New Haven, CT), pRTK-V5/His-Met, pRTK-V5/His-Met-K1110A, pRTK-V5/His-Met-Y1234F/Y1235F, or pRTK-V5/His-Met-Y1349F/Y1354F. Cells were selected and then maintained at 2 and 1 μg/mL puromycin (R&D Systems) or 100 and 50 μg/mL G418, respectively (GIBCO).

Control and MET knockdown lentivirus was produced by cotransfecting HEK 293T cells with pLKO-shRNA constructs (Sigma-Aldrich), pMD2.G, and psPAX2 (Addgene) using lipofectamine (Invitrogen). Supernatants were collected daily for 3 days, combined, and concentrated with Centricon plus-20 filters (Millipore Corporation). Cells were infected overnight in 4 μg/mL polybrene, selected and maintained at 2 and 1 μg/mL puromycin, respectively.

Growth factor stimulation and drug treatment

Cells were stimulated with 10 ng/mL EGF, 50 ng/mL neuregulin (NRG), or 50 ng/mL hepatocyte growth factor (HGF; R&D Systems) for the indicated times. Kinase inhibitors sunitinib, imatinib, nilotinib, tozasertib, gefitinib, U0126, SB203580, dasatinib (LC Laboratories); BMS-754807, AZD-4547, BI2536 (ChemieTek); BMS-536924, MK2206, PLX4032, PD0332991 (Sellek Chemicals); PP2, AR-A014118 (Sigma-Aldrich); Jnk inhibitor II, Syk inhibitor II (EMD Chemicals); PF-573208, CMPD1, BRD7389, SB41542, PHA665752 (Tocris); AZD-7762, NSC625982 (Axon Medchem); CIP-1374 (AlloStem Therapeutics); and PP2 (Calbiochem) were used at 1 μmol/L for the indicated times. Actinomycin D (Sigma) was used at 10 μg/mL, cycloheximide (Sigma) at 10 ng/mL, and bortezomib (LC Laboratories) at 100 nmol/L.

Immunoprecipitation

Cells were lysed in TX100 lysis buffer (20 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, and 1% Triton X-100). Immunoprecipitations were conducted overnight at 4°C with 2 mg protein lysate, 4 μg anti-Met or anti-Cbl antibody (Santa Cruz Biotechnology), and Protein A/G Ultralink Resin (Invitrogen), washed three times in TX100 buffer, and resuspended in 2× Laemmli sample buffer.

Immunoblotting

Cell lysates were prepared in 2× sample buffer or NETN lysis buffer [150 mmol/L NaCl, 1 mmol/L EDTA, 50 mmol/L Tris–HCl pH 7.8, 1% NP40 substitute (Fluka)]. Immunoblots on polyvinylidene difluoride (PVDF) were blocked in 5% nonfat milk/PBST (Dulbecco's PBS, 0.1% Tween-20). Antibodies against phospho-EGFR-Y1068, phospho-ERBB3-Y1197, phospho-MET-Y1003, phospho-MET-Y1234/1235, phospho-Akt-S473, Akt, phospho-p44/42MAPK(Erk1/2)-T202/Y204, p44/42MAPK(Erk1/2), phospho-p38MAPK-T180/Y182, p38MAPK (Cell Signaling Technology); EGFR, ERBB3, MET, ubiquitin, cCBL, GAPDH (Santa Cruz Biotechnology); and V5 (Invitrogen) were incubated overnight at 4°C in 5% milk/PBST. Anti-phospho-tyrosine (Cell Signaling Technology) antibody was incubated 3 hours at room temperature. Membranes were washed in PBST and incubated 1 hour in horseradish peroxidase–conjugated secondary antibodies in 5% milk/PBST.

RNA isolation and real-time PCR

RNA was isolated using the RNeasy Mini Plus Kit with QIAshredder columns (Qiagen) and cDNA was synthesized using the iScript Kit (Bio-Rad). Real-time PCR (RT-PCR) was conducted on a Bio-Rad iCycler RT-PCR machine by combining cDNA, TaqMan Universal Master Mix, and premixed FAM-labeled probes (Applied Biosystems). mRNA quantity was calculated relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the |$2^{ - {\rm \Delta \Delta }C_{\rm t} }$| method.

Migration, invasion, and growth assays

For migration assays, cells were preincubated 3 hours with 10 ng/mL EGF, 1 μmol/L PHA665752, or 1 μmol/L PF-04217903, then plated at 5 × 104 cells per well in 24-well plates with 8-μm filter inserts (BD Biosciences). Conditions were maintained and cells migrated overnight from 0.1% FBS toward 10% FBS. Cells were stained and cell number was averaged from three fields of view per chamber. Invasion assays were conducted similarly but with 1.4 × 105 cells per well in Matrigel invasion chambers (BD Biosciences). For growth assays, cells were plated at 1 × 104 cells per well in a 12-well dish and counted daily for 5 days. Results are the average of at least three replicates and P values were calculated by t test.

Colony formation assay

Cells were plated at 2,000 cells per well in a 6-well dish and grown for 7 days, then washed in PBS, fixed 10 minutes on ice with cold methanol, and stained 2 hours with 0.1% crystal violet/PBS. Cells were rinsed 30 minutes in PBS and air-dried. Colonies were counted using scanned images with ImageJ software.

In vivo metastasis assay

Animal procedures were carried out in accordance with the Yale Institutional Animal Care and Use Committee. A total of 2.5 × 104 cells were suspended in 0.1 mL PBS and injected into the right ventricle of 6-week-old nude athymic mice. Metastasis was detected by bioluminescence with an IVIS Spectrum (Perkin Elmer) as previously described (17). Tumor incidence was quantified by luminescent signal in limbs, spine, or brain in whole body images at given time points and presented as Kaplan–Meier curves. P values were calculated by log-rank test. When possible, animals were sacrificed for harvest of tissues to confirm metastasis by quantifying organ-specific luminescence.

Signaling through EGFR leads to delayed activation of MET

Discrepancies in ERBB–MET interactions among NSCLC cell lines arise from underlying differences in ERBB mutational status and relative expression levels. Because ERBBs cross-activate, it was uncertain which individual ERBB(s) interact with MET or if intermediary signaling pathways indirectly integrate MET with ERBB signaling. To distinguish these possibilities, we used 32D cells that lack ERBBs and MET. Because epithelial cell lines express one or more ERBBs, use of murine hematopoietic 32D cells has been invaluable for elucidating ERBB interactions in reconstruction experiments (18, 19). RT-PCR verified that 32D cells are devoid of EGFR, ERBB2, ERBB3, ERBB4, MET, and agonists EGF, TGF-α, AREG, BTC, NRG1, and HGF, consistent with previous studies (data not shown; refs. 18, 19). We then ectopically expressed EGFR and MET alone or in combination (Fig. 1A).

In 32D cells that are engineered to express human EGFR or MET singly, EGF stimulated EGFR phosphorylation and HGF stimulated MET phosphorylation, as expected (Supplementary Fig. S1A). EGF did not induce MET phosphorylation and HGF did not induce EGFR phosphorylation, verifying specificity of ligands for their receptors (Supplementary Fig. S1A). In cells expressing both EGFR and MET (EGFR/MET), HGF failed to induce phosphorylation of EGFR at Y1068 (Supplementary Fig. S1B), however, EGF increased MET phosphorylation and protein levels (Fig. 1B). Although EGF induces rapid phosphorylation of EGFR, EGF-induced phosphorylation of MET was delayed, occurring after 2 hours and increasing through 10 hours (Fig. 1B). EGF stimulation induced an increase in total MET levels without affecting the abundance of MET mRNA (Fig. 1C). Also, quantification of phospho-MET to total MET revealed increased stoichiometry of MET phosphorylation induced by EGF (Fig. 1D). Overall, EGFR upregulates MET abundance and relative Tyr phosphorylation over an extended time frame, and does not require other ERBB family receptors.

EGFR and MET kinases are required for EGF-dependent activation of MET

EGF-induced MET phosphorylation could occur through direct phosphorylation of MET by EGFR, EGF-dependent activation of MET autophosphorylation, or another intermediary kinase. To investigate the importance of kinase activity in this receptor interaction, small-molecule TKIs gefitinib and PHA665752 were used to inhibit EGFR and MET, respectively. Specificity of inhibitors for the cognate receptors was verified (Supplementary Fig. S2A and S2B). Although slight changes in phospho-EGFR were seen with single inhibitors, these changes reflected similar differences in total protein level and were not seen consistently (Fig. 1E). Treatment of EGFR/MET 32D cells with gefitinib prevents EGF-induced MET phosphorylation, while PHA665752 completely abolishes both EGF-induced and basal phosphorylation of MET (Fig. 1E). Similarly, EGF was unable to induce robust phosphorylation of kinase-inactive MET mutants K1110A and Y1234F/Y1235F, while still activating wild-type MET receptor and docking site-mutant Y1349F/Y1354F (Supplementary Fig. S2C). Hence, EGF-induced MET phosphorylation in 32D cells requires activity of both EGFR and MET kinases.

Enhanced MET signaling is one mechanism by which NSCLC patients with EGFR-activating mutations, such as EGFR L858R, become resistant to therapy with the EGFR inhibitor erlotinib (2–4). Recent evidence suggests that MET and activated EGFR also interact in drug-naïve lung cancer cells independent of drug-resistance mechanisms (6–9, 12). To determine if activated EGFR alleles commonly found in NSCLC activate MET, MET was expressed in 32D cells in combination with EGFR L858R. Similar to wild-type EGFR, EGFR L858R induced MET phosphorylation after 4 hours of EGF stimulation, and activation was prevented by gefitinib (Fig. 1F). These results show for the first time that EGFR activates MET independent of other ERBB family members, and that both wild-type and activated EGFR induce MET phosphorylation.

ERBB3 enhances EGFR-induced MET phosphorylation

ERBB3 is nearly devoid of intrinsic catalytic activity, and functions through heteromers with other ERBBs (20, 21). Although ERBB3 phosphorylation can be dependent on MET in gefitinib-resistant NSCLC cells, it is unclear whether ERBB3 and MET interact in drug-naïve cells or if another ERBB family member is required (1). To investigate signaling interactions between ERBB3 and MET, ERBB3 was coexpressed with MET alone or with EGFR/MET in 32D cells. Coexpression of ERBB3 enhanced EGF-induced MET phosphorylation compared with EGFR/MET alone (Fig. 2A). Similar to EGFR/MET cells, stimulation of EGFR/ERBB3/MET cells with ERBB ligands did not affect MET mRNA levels (Fig. 2B). Increased EGF-dependent phospho-MET relative to total MET occurs within 4 hours in EGFR/ERBB3/MET cells and is maximal by 7 hours (Fig. 2C). Hence, ERBB3 enhances EGF-induced MET phosphorylation, likely through signal amplification and broadening by EGFR/ERBB3 heteromers. In fact, coexpression of EGFR and ERBB3 alters EGF-induced downstream signaling, leading to decreased phospho-Akt, increased phospho-Erk, and possibly increased total extracellular signal–regulated kinase (Erk; Fig. 2D).

Having determined that ERBB3 alters signaling to MET by EGFR, we investigated whether MET can be activated by ERBB3 alone. Stimulation of ERBB3/MET cells with the ERBB3 ligand NRG1 for up to 10 hours failed to activate MET or Akt (Fig. 2E). Lack of ERBB3/MET signaling activity suggests that these receptors cannot act as direct dimerization partners. However, when EGFR is coexpressed in ERBB3/MET cells, NRG induces MET phosphorylation (Fig. 2E), indicating that ERBB3 can induce MET phosphorylation when it has a catalytically active signaling partner. Therefore, MET cannot act as a functional dimerization partner for ERBB3, but ligand-activated ERBB3 is able to induce MET phosphorylation when coexpressed with EGFR.

EGFR activates MET through protein stability and MAPK signaling in 32D cells

EGFR may activate MET through increased autocrine MET signaling by increasing transcription or stability of MET or HGF. EGF-induced MET activation is actinomycin D–sensitive and thus requires transcription (Supplementary Fig. S3A). However, MET activation was not associated with changes in MET mRNA levels (Fig. 1C) and HGF mRNA was not expressed (data not shown). Therefore, transcriptional dependency in 32D cells involves another component of the pathway.

The changes observed in MET levels without an increase in MET mRNA suggest instead that EGFR stabilizes MET protein. 32D cells expressing MET or EGFR/MET were treated with the protein synthesis inhibitor, cycloheximide, and the half-life of MET protein was evaluated. EGF failed to stabilize MET in cells expressing MET alone, as expected (Fig. 3A). However, MET was stabilized by coexpression of EGFR and further stabilized when EGFR was activated by EGF (Fig. 3A). Overexpression of EGFR increased the MET half-life from 3.5 to 5.5 hours, whereas EGF-stimulation of EGFR further increased the half-life to 6.3 hours (Fig. 3B). It is likely that the increase in MET half-life by EGFR expression is caused by basal EGFR activity from high expression levels. Overall, MET protein is stabilized by EGFR signaling in 32D cells.

In addition to regulating MET protein stability, it is possible that EGFR activates MET through signaling proteins such as Src, which acts as an intermediary in EGFR-induced MET phosphorylation of the EGFR wild-type NSCLC cell line, 201T (12). To determine if this is a general mechanism of EGFR-induced MET phosphorylation, Src inhibitors were evaluated for the ability to prevent EGFR–MET cross-talk in 32D cells. Interestingly, neither dasatinib nor PP2 prevented EGF-induced MET phosphorylation (Fig. 3C). To confirm this result, MET was expressed in SYF−/− MEFs with constitutional knockout of Src family members Src, Yes, and Fyn (16). EGF stimulation led to turnover of EGFR, causing decreased EGFR protein, however, activated EGFR was still detectable, indicating that EGFR signaling does occur (Supplementary Fig. S3B). Despite the lack of Src proteins, EGF activated MET accumulation and phosphorylation, indicating Src family proteins are not absolutely required for EGF-induced MET phosphorylation (Supplementary Fig. S3B). Thus, although Src is involved in EGFR-induced MET activation in some contexts, it is not a unique mediator of and is not required for EGFR–MET cross-talk.

To identify intermediaries in EGF-induced MET phosphorylation in 32D cells, pools of protein kinase inhibitors were tested for interference with this process (Supplementary Fig. S3C). Of these, inhibitor pools A, C, and D prevented EGF-induced MET phosphorylation. Pool C yielded inconsistent results among experiments, and only partially inhibited Met phosphorylation. Pool A includes at least one agent, BMS754807, which is now reported to have some inhibitory activity on MET. For these reasons, we focused on pool D, composed of mitogen-activated protein kinase (MAPK) signaling inhibitors.

To evaluate the role of MAPK in EGFR-MET signaling, inhibitors of MEK1/2 and p38MAPK, U0126 and SB203580, were tested for ability to prevent EGFR-MET crosstalk. These inhibitors had moderate or no effect on EGF-induced MET phosphorylation singly, but nearly completely prevent it when combined (Fig. 3D). This suggests that EGF-induced MET phosphorylation works through the RAS–ERK–p38MAPK pathway, and compensation occurs through parallel MAPK cascades when only one is inhibited.

Although gefitinib inhibits EGF-induced MET phosphorylation in cells with wild-type EGFR or EGFR L858R (Fig. 1E and F), it is ineffective against the resistance mutation, T790M. To determine if MEK and p38MAPK inhibitors prevent EGFR activation of MET in cells with T790M mutations, EGFR L858R/T790M (LT) receptors were coexpressed with MET in 32D cells. Gefitinib did not affect EGFR or MET phosphorylation in these cells, but the combination of U0126/SB203580 substantially decreased MET phosphorylation (Fig. 3E). Hence, MEK/p38MAPK inhibitors can inhibit EGFR–MET signaling in cells where gefitinib is not effective. Overall, EGFR activation of MET in 32D cells occurs through increased stability of MET protein and intermediary signaling through MAPK.

EGFR regulates MET at multiple levels in NSCLC cell lines

To confirm the presence of EGFR–MET cross-talk in NSCLC cell lines, the EGFR-mutant cell line, HCC827, was treated with gefitinib for up to 24 hours. Gefitinib treatment induced rapid decreases in MET phosphorylation and delayed decreases in MET protein levels (Fig. 4A). These different rates of change suggest that EGFR can affect MET phosphorylation independent of MET protein levels. Unlike 32D cells, EGFR signaling in HCC827 cells regulates MET protein and MET mRNA with similar kinetics following gefitinib treatment (Fig. 4B and C), emphasizing the complex dynamics of EGFR–MET signaling in NSCLC cell lines. Similar changes in MET phosphorylation, protein, and mRNA were observed following gefitinib treatment of another EGFR-mutant cell line, PC9 (Supplementary Fig. S4).

Although changes in MET protein levels following gefitinib treatment may be caused by decreased mRNA abundance, there is evidence for EGFR-dependent modulation of MET at the protein level through MET ubiquitination. Following stimulation by HGF, MET is ubiquitinated by the E3 ligase, c-CBL, and internalized for intracellular trafficking or degradation (22–25). Although three cell lines with wild-type EGFR underwent robust MET ubiquitination following ligand stimulation, three of four cell lines with EGFR-activating mutations did not (Fig. 4D). Importantly, MET ubiquitination was associated with phosphorylation of c-CBL (Fig. 4D). This corroborates our finding that, in 32D cells, EGFR stabilizes MET protein levels independent of transcriptional changes and indicates that EGFR modulates MET at multiple levels in NSCLC cell lines.

To determine if intermediary signaling cross-talk occurs through MAPK in NSCLC cell lines, HCC827 cells were treated with U0126/SB203580. Interestingly, treatment with either gefitinib or U0126/SB203580 caused a reduction in total MET levels, accompanied by elimination of phospho-MET (Fig. 4E). This confirms that signaling observed in 32D cells resembles NSCLC cell lines and that MEK/p38MAPK pathways act as signaling intermediaries between EGFR and MET in multiple cell types. Although NSCLC cells display a more complicated relationship between EGFR and MET than 32D cells, NSCLC cells similarly show independent regulation of MET protein and phosphorylation by EGFR signaling. Importantly, the existence of EGFR–MET cross-talk in multiple NSCLC cell lines indicates that EGFR-induced MET activation may have a biologic significance.

MET regulates EGFR-induced migration and invasion in NSCLC cells

MET regulates many cellular phenotypes, including cell growth and motility. We compared EGFR-only with EGFR/MET 32D cells and found that EGFR–MET cross-talk had no effect on cell viability (data not shown). Because MET signaling is also important for cell motility, it is possible that EGFR activation of MET modulates this phenotype. MET is required for EGF-induced cell invasion and motility in EGFR wild-type NSCLC cells but it was unclear if this is true in cells with EGFR-activating mutations (12). Hence, we confirmed the previously reported MET-dependence for EGF-induced migration and invasion in the EGFR wild-type NSCLC cell line A549 (Supplementary Fig. S5A) and tested additional cell lines with various EGFR mutations. Indeed, PHA665752 prevented EGF-induced migration of NSCLC cell lines with both wild-type and activated EGFR (Fig. 5A). Similarly, MET inhibition prevented EGF-induced invasion through Matrigel of both EGFR wild-type and mutant NSCLC cells (Fig. 5B). This was confirmed with an additional highly specific MET inhibitor, PF-04217903, which has strong activity in NSCLC cells (Supplementary Fig. S5B and S5C). Therefore, EGFR–MET signaling is active in NSCLC cell lines with multiple EGFR-activating mutations and mediates aggressive phenotypes including migration and invasion.

EGFR–MET signaling is important for metastatic behavior

MET promotes EGF-induced NSCLC cell invasion, so it may have a role in the biology of metastatic lung cancer cells addicted to EGFR. To investigate this, we compared EGFR–MET signaling in an EGFR-mutant NSCLC cell line, PC9, and its metastatic subpopulation, PC9-BrM3 (15, 26, 27). Both parental PC9 and PC9-BrM3 cell lines harbor constitutively active EGFR Δ746–750 and are dependent on EGFR signaling for survival. However, PC9-BrM3 cells have a marked increase in the capacity to invade and colonize distant organs, most notably the brain (15).

EGFR inhibition in both PC9 and PC9-BrM3 cells decreases MET phosphorylation and protein levels, confirming the presence of EGFR–MET signaling in these cells (Fig. 6A). However, the mechanism of EGFR–MET signaling differs, as MEK/p38MAPK inhibition reduces MET phosphorylation and protein levels in PC9-BrM3 cells, but has little effect on PC9 cells (Fig. 6A). Different requirements for MAPK in EGFR–MET signaling may correlate with lower Erk phosphorylation observed in PC9 compared with PC9-BrM3 cells (Fig. 6B). In addition, mRNA levels of both EGFR and MET trended higher in PC9-BrM3 than PC9 cells (Fig. 6C). Although possibly coincidental, these differences in EGFR–MET signaling raised the possibility that this pathway contributes to the highly metastatic behavior of PC9-BrM3 cells.

To directly evaluate the role of MET in EGFR-driven phenotypes of PC9 and PC9-BrM3 cells, a lentiviral system was used to induce MET knockdown (Supplementary Fig. S6A). Similar to growth studies in 32D cells, MET knockdown did not affect growth of PC9 or PC9-BrM3 cells (Fig. 6D). In contrast with other NCSLC cells, MET was not required for EGF-induced cell invasion of parental PC9 cells and knockdown inconsistently affected cell migration (Fig. 6E). Interestingly, MET knockdown prevented both EGF-induced cell migration and invasion in PC9-BrM3 cells (Fig. 6E). This was confirmed with PHA665752 treatment, showing consistent cellular responses to MET inhibition and knockdown (Supplementary Fig. S6B). The role of MET in clonogenic potential also differed between the cell lines. MET knockdown with two different short hairpin RNAs (shRNA) yielded inconsistent changes in clonogenic colony formation of PC9 cells, suggesting these effects were not MET specific (Fig. 6F). However, knockdown of MET in PC9-BrM3 cells consistently reduced clonogenic colony formation (Fig. 6F). Although EGFR–MET cross-talk exists in parental PC9 cells, the functional consequence of EGFR–MET cross-talk is different in metastatic cells where it enhances clonogenicity and invasion, independently of cell survival. Thus, although NSCLC cells generally require mutant EGFR signaling for tumorigenesis, cross-talk with MET may further enhance metastatic progression.

MET signaling modulates metastasis in many epithelial cancers (28–31). In patients with NSCLC, MET expression and phosphorylation correlate with the incidence of brain metastasis, but the functions of MET in lung cancer metastasis remain uncharacterized (32). A rate-limiting step in metastasis is invasion and colonization of distant organs by cancer cells following dissemination into the bloodstream (33). To assess effects of EGFR–MET signaling on metastatic colonization, we injected luciferase-marked PC9-BrM3 cells into the arterial circulation of immunocompromised mice. Incidence and burden of metastasis were compared following intracardiac injection of PC9-BrM3 cells stably expressing control or MET knockdown vectors. Surprisingly, no significant difference was seen in overall tumor burden between control and MET knockdown (Fig. 7A). Although MET knockdown had no effect on bone metastasis, incidence of brain metastasis was significantly delayed and/or attenuated (Fig. 7B and C). When possible, animals were sacrificed to confirm metastasis by brain imaging. Although there was no significant difference in the brain tumor burden in animals that harbor metastasis at endpoint, MET knockdown decreased the frequency of brain metastasis from 100% to 30% (Fig. 7B). This corroborates in vitro results of MET knockdown decreasing cell invasion but not growth (Fig. 6). In summary, MET signaling enhances in vivo brain metastatic invasion by EGFR-addicted NSCLC cells. This supports the clinical correlation observed between MET expression and metastatic relapse in patients with NSCLC, and emphasizes the significance of EGFR–MET signaling in aggressive stages of lung cancer progression (32).

Existence of signaling interactions between MET and the ERBBs is well established, but mechanism(s) are incompletely understood. For reconstruction of defined sets of receptors, it was essential to use a receptor-null cell background, which was not possible for epithelial lines as they express one or more ERBBs. We used murine 32D cells, which do not express endogenous MET or any ERBBs, to investigate interactions between MET and individual ERBBs. EGFR signaling was sufficient to induce MET phosphorylation and increase MET protein levels, but MET activation was augmented by coexpression of ERBB3. Rather than direct cross-phosphorylation, EGFR activation of MET occurs through combined stabilization of MET protein and intermediary signaling through MAPK. EGFR–MET cross-talk in lung cancer cells is more complex, and involves multiple levels of regulation including RNA, protein, and phosphorylation. Significantly, in lung carcinoma, EGFR–MET signaling is exaggerated in metastatic cells where MET enhances EGFR-mediated aggressive phenotypes including migration, invasion, and metastasis, independently of cell growth.

EGFR, ERBB2, and ERBB3 have all been implicated in ERBB–MET signaling cross-talk. However, promiscuous ERBB dimerization and coexpression of multiple ERBBs in NSCLC cells makes it difficult to determine which ERBB(s) activate MET. Using a model system, we determined that EGFR activation by ligand or mutation is sufficient to induce MET phosphorylation (Fig. 1). Although we did not observe activation of EGFR by MET, only one EGFR phosphorylation site was tested and we cannot rule out such cross-talk entirely. In addition, ERBB3 enhances EGFR-driven phosphorylation of MET and activates MET itself when part of an active dimer (Fig. 2). Consequently, MET activation may occur in NSCLC tumors with active EGFR but could be enhanced in tumors coexpressing other ERBB receptors. In addition to enhancing EGFR-induced MET activation, ERBB3 alters downstream signaling of EGFR through MAPK and phosphoinositide 3-kinase (PI3K) pathways (Fig. 2). Therefore, coexpression of ERBB3 in NSCLC may affect not only ERBB–MET cross-talk but intracellular signaling as well.

Multiple different mechanisms may regulate EGFR activation of MET including direct receptor interactions, regulation of MET autocrine signaling, or indirect signaling through intermediary proteins. We have excluded direct receptor cross-phosphorylation based on delayed EGF-induced MET phosphorylation, requirement for both EGFR and MET kinase activities, and the inability of MET to act as a dimerization partner for ERBB3 (Figs. 1 and 2). Instead, we see evidence for ERBB regulation of MET through both autocrine and intermediary signaling.

In some cell lines, EGFR activates MET by inducing MET transcription, thereby increasing the concentration of MET at the membrane, leading to more receptor collisions, homodimer formation, and MET activation (12, 34, 35). In our model system, EGFR regulates MET at the protein level by extending the MET half-life (Fig. 3), which would also increase MET availability and enhance autocrine activation. In papillary renal carcinoma, MET mutations lead to increased recycling and decreased degradation, causing increased activation of MET that drives tumorigenesis (36). We see decreased MET ubiquitination in EGFR-mutant NSCLC cells, which would similarly alter MET recycling and degradation (Fig. 4). Therefore, active EGFR in NSCLC tumors may regulate MET through altered receptor trafficking to enhance already robust tumorigenic signaling.

Dulak and colleagues reported that Src is required for EGFR-induced MET activation independent of MET transcription, indicating MET can also be regulated through intermediary signaling pathways (12). We found that MAPK signaling correlated with higher ERBB-driven MET phosphorylation and was required for EGFR-dependent MET activation in cells with both wild-type and mutant EGFR (Figs. 3 and 4). Different requirements for Src and MAPK signaling may depend on the cell line, and it is possible that both pathways act as intermediates in different contexts. In addition, Src can enhance activation of MAPK in cancer cells directly or by binding to EGFR, so these pathways may have additive functions (37). Investigation of additional experimental models and tumor samples could reveal the relative roles of these proteins in EGFR–MET cross-talk in different cell types and contexts.

Existence of EGFR–MET signaling in multiple normal and cancer cell lines suggests that MET modulates biologic outcomes of EGFR signaling. Although both receptors activate PI3K/Akt signaling, MET was not required for cell survival in 32D or NSCLC cells. EGFR and MET also commonly regulate cell motility and MET promotes EGF-induced cell motility and invasion in EGFR wild-type NSCLC cells (8, 12). NSCLC cells with EGFR mutations have constitutively hyperactive EGFR signaling and it was unclear if MET could regulate EGFR-driven phenotypes in these cells. We found, however, that MET facilitates EGF-induced migration and invasion in EGFR-mutant NSCLC cells (Fig. 5), showing that MET signaling enhances other aggressive phenotypes of EGFR-mutant lung cancers.

Moreover, we discovered differences in EGFR–MET cross-talk in NSCLC cell lines of varying metastatic potential. In metastatic PC9-BrM3 derivatives, but not parental PC9 cells, EGFR–MET signaling enhanced migration, invasion, and colony formation, and was dependent on MAPK signaling (Fig. 6). Differences between PC9 and PC9-BrM3 cells reveal context-dependent requirements for EGFR–MET signaling in metastatic NSCLC cells. Because PC9-BrM3 cells were selected to be highly metastatic in vivo, enhanced signaling in these cells suggests EGFR–MET cross-talk could be a mediator of metastasis and that the mechanism of cross-talk can change throughout progression.

MET regulates metastasis of gastric cancer, renal papillary carcinomas, and breast cancer and MET expression and activity in NSCLC correlate with occurrence of brain metastasis (29, 32, 38, 39). In addition, MET copy number, expression, and phosphorylation are enriched in NSCLC brain metastases compared with primary tumors (32). Despite association of MET with metastasis, a direct role for MET in metastasis of NSCLC has not been previously identified. We observed that MET knockdown in highly metastatic NSCLC PC9-BrM3 cells caused significant reduction in metastasis to the brain (Fig. 7). Differences between brain and other organ sites may depend on the brain being a more stringent organ to invade, which is supported by delayed detection of brain metastasis versus other sites in our model. In addition, differences in brain tumor incidence but not burden suggest that an important role of MET in lung cancer metastasis may be to promote invasion of the brain environment. The significance of this result is underscored by the fact that the brain is the major metastatic site and source of morbidity in patients with lung cancer (40). The inability of mouse HGF to activate human MET makes it difficult to address the relative contributions of EGFR–MET cross-talk versus HGF paracrine MET activation toward metastasis with this model, but does not detract from the importance of MET in promoting NSCLC progression. These results support further investigation of the role of MET in EGFR-mutant NSCLC brain metastasis and the use of MET inhibitors to prevent metastatic progression of NSCLC in patients.

Previous clinical trials for MET inhibitors in NSCLC were focused on patients who were resistant to EGFR TKIs due to MET amplification. The ability of both wild-type and mutant EGFR to activate MET, as well as MET regulation of EGFR-driven migration, invasion, and metastasis, suggests that MET inhibitors may be beneficial to patients with varying mutational status. In fact, a recent clinical trial of combination EGFR-MET inhibitors that did not prescreen patients for mutation status found increased progression free and overall survival in patients with wild-type EGFR NSCLC (41). In addition, EGFR–MET signaling becomes more important at later stages of NSCLC progression, indicating that use of MET inhibitors in patients with early-stage cancer may prevent occurrence of invasion and metastasis and should be evaluated as a therapeutic option. Further investigation of EGFR–MET cross-talk will be useful for determining which patients may benefit most from combination therapies and for identifying potential new targets to prevent EGFR-induced MET activation.

No potential conflicts of interest were disclosed.

Conception and design: J.L. Breindel, D.F. Stern

Development of methodology: J.L. Breindel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.W. Haskins, E.P. Cowell, M. Zhao, D.X. Nguyen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.L. Breindel, E.P. Cowell, D.X. Nguyen, D.F. Stern

Writing, review, and/or revision of the manuscript: J.L. Breindel, J.W. Haskins, E.P. Cowell, D.X. Nguyen, D.F. Stern

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Zhao

Study supervision: D.F. Stern

The authors thank Drs. A. Koleske, D. DiMaio, and S. Agarwal for cell lines, N. Hynes for plasmids and K. Politi for plasmids and advice about EGFR signaling in lung cancer.

This work was funded in part by the DOD CDMRP Breast Cancer Research Program #W81XWH-08-1-0780 (J.L. Breindel), CMB training grant #T32 GM007223 (J.W. Haskins), USPHS grant R01CA45708 (D.F. Stern), and Uniting Against Lung Cancer (D.X. Nguyen). D.X. Nguyen is a scholar of the V Foundation for Cancer Research, Yale Center for Clinical Investigation, and Young Investigator of the International Association for the Study of Lung Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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