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Inducible EGFR T790M-Mediated Gefitinib Resistance in Non-small Cell Lung Cancer Cells Does Not Modulate Sensitivity to PI103 Provoked Autophagy

      Introduction

      Non-small cell lung cancer (NSCLC) with certain activating mutations in the epidermal growth factor receptor (EGFR) is sensitive to the small molecule EGFR tyrosine kinase inhibitors gefitinib and erlotinib, although acquired resistance eventually develops. Resistance is often mediated by acquisition of the T790M mutation in the activated EGFR allele. The aim of this study was to investigate in an EGFR tyrosine kinase inhibitor sensitive NSCLC cell line model, the impact of induced EGFR T790M expression on the cell biology and sensitivity to novel therapeutic strategies.

      Methods

      Doxycycline inducible EGFR T790M-mediated drug resistance was generated in the clinically relevant HCC827 NSCLC cell line. Cell fate, the activities of EGFR and downstream signaling molecules, and the sensitivity to downstream inhibition of EGFR signaling networks were examined in the presence or absence of induced EGFR T790M expression.

      Results

      Inducible EGFR T790M expression generated acquired resistance to EGFR inhibitors in HCC827 cells as expected. However, induced EGFR T790M expression did not affect activity of EGFR downstream signaling pathways or cell proliferation under the conditions tested. Moreover, sensitivity to inhibition of signaling molecules downstream of EGFR was unaffected by induced EGFR T790M. Importantly, HCC827 cells remained sensitive to class I phosphatidyl-inositol-3-kinase and mammalian target of rapamycin inhibition, which provoked pronounced autophagy, without significant apoptosis.

      Conclusions

      Phosphatidyl-inositol-3-kinase /mammalian target of rapamycin inhibition is a potentially effective therapeutic strategy against NSCLC with acquired resistance to EGFR inhibition. However, the implications of drug-induced autophagy in NSCLC need further exploration.

      Key Words

      Epidermal growth factor receptor (EGFR) activity is frequently deregulated in solid tumors affecting signaling networks important for tumor formation, maintenance, and development. This is mediated through activation of downstream signaling components including protein kinase B (PKB or Akt)/phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK)/ERK kinase (MEK).
      • Yarden Y
      • Sliwkowski MX
      Untangling the ErbB signalling network.
      Patients with non-small cell lung cancer (NSCLC) whose tumors harbor somatic and activation mutations in EGFR exons 18–21 show dramatic clinical responses to reversible EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib.
      • Lynch TJ
      • Bell DW
      • Sordella R
      • et al.
      Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.
      • Paez JG
      • Janne PA
      • Lee JC
      • et al.
      EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.
      • Pao W
      • Miller V
      • Zakowski M
      • et al.
      EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib.
      Most prevalent of these are a single amino acid substitution L858R and short in-frame deletions in exon 19.
      • Sharma SV
      • Bell DW
      • Settleman J
      • et al.
      Epidermal growth factor receptor mutations in lung cancer.
      These mutations are more common in females with adenocarcinoma classed as “never-smokers.”
      • Sharma SV
      • Bell DW
      • Settleman J
      • et al.
      Epidermal growth factor receptor mutations in lung cancer.
      Despite initial response, patients with NSCLC with activating EGFR mutations develop resistance to EGFR TKIs.
      • Engelman JA
      • Janne PA
      Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.
      Approximately half of acquired resistance cases are accounted for by acquisition of an EGFR T790M mutation, which occurs in cis with the exons 18–21 activating mutation.
      • Kobayashi S
      • Boggon TJ
      • Dayaram T
      • et al.
      EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.
      • Pao W
      • Miller VA
      • Politi KA
      • et al.
      Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.
      MET amplification is the second most prevalent event identified in this patient population,
      • Bean J
      • Brennan C
      • Shih JY
      • et al.
      MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.
      • Engelman JA
      • Zejnullahu K
      • Mitsudomi T
      • et al.
      MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.
      and rare EGFR mutations L747S, D761Y, and T854A also decrease sensitivity to gefitinib and erlotinib.
      • Balak MN
      • Gong Y
      • Riely GJ
      • et al.
      Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.
      • Costa DB
      • Halmos B
      • Kumar A
      • et al.
      BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations.
      • Bean J
      • Riely GJ
      • Balak M
      • et al.
      Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma.
      EGFR T790M mutation mediated resistance to gefitinib and erlotinib has been confirmed in vitro and in mouse models.
      • Blencke S
      • Zech B
      • Engkvist O
      • et al.
      Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors.
      • Regales L
      • Balak MN
      • Gong Y
      • et al.
      Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.
      • Godin-Heymann N
      • Bryant I
      • Rivera MN
      • et al.
      Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
      • Li D
      • Shimamura T
      • Ji H
      • et al.
      Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.
      • Vikis H
      • Sato M
      • James M
      • et al.
      EGFR-T790M is a rare lung cancer susceptibility allele with enhanced kinase activity.
      The target site for EGFR TKIs is the T790 residue in EGFR located at the back of the adenosine triphosphate (ATP) binding cleft. Mutation of this so-called “gatekeeper” residue to a bulkier methionine was believed to sterically hinder TKI binding.
      • Kobayashi S
      • Boggon TJ
      • Dayaram T
      • et al.
      EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.
      However, in vitro, T790M mutation increases affinity of activated EGFR mutants to ATP, while having a modest negative effect on drug affinity for EGFR.
      • Yun CH
      • Mengwasser KE
      • Toms AV
      • et al.
      The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.
      These results implicate competitive inhibition in favor of ATP (rather than steric hindrance) as the major contributor to T790M-mediated drug resistance.
      • Yun CH
      • Mengwasser KE
      • Toms AV
      • et al.
      The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.
      Rare familial cases of lung cancer are associated with germline T790M mutation in EGFR,
      • Bell DW
      • Gore I
      • Okimoto RA
      • et al.
      Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR.
      and introduction of T790M mutation into wild-type EGFR provided modest increase in catalytic activity
      • Yun CH
      • Mengwasser KE
      • Toms AV
      • et al.
      The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.
      and enhanced its oncogenic potential.
      • Vikis H
      • Sato M
      • James M
      • et al.
      EGFR-T790M is a rare lung cancer susceptibility allele with enhanced kinase activity.
      In contrast, introduction of T790M mutation on activated (L858R) EGFR decreased catalytic activity.
      • Yun CH
      • Mengwasser KE
      • Toms AV
      • et al.
      The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.
      Despite these results, when constitutively or transiently expressed in model cell lines, T790M-containing double mutants of EGFR display enhanced oncogenic properties compared with single mutants.
      • Godin-Heymann N
      • Bryant I
      • Rivera MN
      • et al.
      Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
      • Engelman JA
      • Mukohara T
      • Zejnullahu K
      • et al.
      Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer.
      • Mulloy R
      • Ferrand A
      • Kim Y
      • et al.
      Epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib.
      Although these effects should be mediated by enhanced activation of downstream signaling pathways, this was not observed in all cell lines.
      • Godin-Heymann N
      • Bryant I
      • Rivera MN
      • et al.
      Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
      • Shimamura T
      • Li D
      • Ji H
      • et al.
      Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance.
      Although cells expressing double mutants with EGFR T790M are resistant to reversible EGFR TKIs, they retain sensitivity to high concentrations of irreversible EGFR inhibitors.
      • Godin-Heymann N
      • Ulkus L
      • Brannigan BW
      • et al.
      The T790M “gatekeeper” mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor.
      The latter demonstrated limited activity in mouse models of EGFR T790M-expressing lung cancer and against clinical NSCLC with acquired resistance to gefitinib or erlotinib.
      • Li D
      • Shimamura T
      • Ji H
      • et al.
      Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.
      • Wong KK
      HKI-272 in non small cell lung cancer.
      Combination of the irreversible EGFR inhibitor HKI-272 with mammalian target of rapamycin (mTOR) inhibitor rapamycin or Hsp90 inhibition has shown promise in preclinical models against T790M-mediated drug resistance.
      • Regales L
      • Balak MN
      • Gong Y
      • et al.
      Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.
      • Li D
      • Shimamura T
      • Ji H
      • et al.
      Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.
      • Shimamura T
      • Li D
      • Ji H
      • et al.
      Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance.
      Another probable route to overcoming EGFR T790M-mediated drug resistance would be direct pharmacological inhibition of PI3K or MEK, i.e., vertical attack on the EGFR pathway. MEK inhibition induces changes in the proapoptotic protein BIMEL identical to those observed during gefitinib/erlotinib induced apoptosis in NSCLC cells sensitive to these agents.
      • Costa DB
      • Halmos B
      • Kumar A
      • et al.
      BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations.
      • Cragg MS
      • Kuroda J
      • Puthalakath H
      • et al.
      Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
      • Gong Y
      • Somwar R
      • Politi K
      • et al.
      Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas.
      However, NSCLC cells expressing activated EGFR mutants were insensitive to MEK inhibitor PD0325901.
      • Cragg MS
      • Kuroda J
      • Puthalakath H
      • et al.
      Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
      • Gong Y
      • Somwar R
      • Politi K
      • et al.
      Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas.
      • Pratilas CA
      • Hanrahan AJ
      • Halilovic E
      • et al.
      Genetic predictors of MEK dependence in non-small cell lung cancer.
      Class I PI3Ks are also activated downstream of ligand-bound EGFR, resulting in activation of PKB/Akt and mTOR.
      • Yuan TL
      • Cantley LC
      PI3K pathway alterations in cancer: variations on a theme.
      In contrast to MEK inhibition, treatment with the pan-PI3K inhibitors LY294002 and wortmannin did not elicit apoptosis-associated changes in BIMEL in NSCLC cells sensitive to EGFR TKIs. This was perhaps surprising, because inhibition of PI3K/Akt signaling has been correlated with decreased cell survival.
      • Erhardt P
      • Cooper GM
      Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL.
      However, this concept relies mainly on experiments using PI3K inhibitors with relatively broad selectivity. Recently, inhibitors targeted more specifically to different classes of PI3K have been developed. One example, PI103 inhibits class I PI3K and mTOR and showed promising preclinical activity against malignant glioma.
      • Fan QW
      • Knight ZA
      • Goldenberg DD
      • et al.
      A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.
      • Raynaud FI
      • Eccles S
      • Clarke PA
      • et al.
      Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.
      Treatment of different cancer cell lines with PI103 does not always result in apoptosis but often induces cell cycle arrest.
      • Fan QW
      • Knight ZA
      • Goldenberg DD
      • et al.
      A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.
      • Raynaud FI
      • Eccles S
      • Clarke PA
      • et al.
      Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.
      PI103 was also shown to induce autophagy in U87MG and PC3 cancer cell lines.
      • Degtyarev M
      • De Maziere A
      • Orr C
      • et al.
      Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
      Autophagy, (“self-eating” stimulated by unfavorable microenvironments), is negatively regulated by Class I PI3Ks, through activation of Akt and mTOR.
      • Gozuacik D
      • Kimchi A
      Autophagy and cell death.
      Although a cell survival role for autophagy has been established, autophagy-dependent cell death has also been described as “type II programmed cell death.”
      • Hoyer-Hansen M
      • Jaattela M
      Autophagy: an emerging target for cancer therapy.
      Once autophagy is initiated, a subsequent block of late stages in the process might drive cells into apoptosis.
      • Degtyarev M
      • De Maziere A
      • Orr C
      • et al.
      Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
      To study EGFR T790M activity and drug sensitivity in a clinically relevant cell line, inducible expression of an activated EGFR mutant containing the T790M mutation was generated in EGFR TKI sensitive HCC827 NSCLC cells. This is, to our knowledge, the first report of inducible expression of EGFR T790M in a relevant cell line. Inducible systems have the advantage of being truly isogenic, allowing specific effects of the expressed gene to be dissected from underlying genetic differences between control and expressing cells. The system of inducible resistance to EGFR TKIs described here was used to study the impact of EGFR T790M expression on cell proliferation, the activation of signaling pathways downstream of EGFR, and the resultant sensitivity to inhibition of MEK, PI3K, and mTOR. The ability of PI3K and mTOR inhibition to induce cytostasis, apoptosis, or autophagy in this cellular context was evaluated, and the implications of autophagy induction in the clinic will be discussed.

      MATERIALS AND METHODS

      Drugs and Antibodies

      Gefitinib (Iressa) was obtained from AstraZeneca (Alderley Park, UK). PI103, U0126, PD98059, and CL-387785 were from Calbiochem. All drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Doxycycline (Invitrogen, Paisly, UK) was dissolved in H2O, stored at −20°C, and used at 100 ng/ml unless stated otherwise. Antibodies against the following proteins were from cell signaling: Akt, phospho-Ser-473-Akt, phospho-EGFR (all phosphorylated residues), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204); p70 S6-kinase and phospho-Thr389-p70 S6 kinase and poly ADP ribose polymerase (PARP). The following antibodies were sourced as follows; EGFR (Santa Cruz, Santa Cruz, CA), BIM (Calbiochem, Darmstadt, Germany), LC3 (Nanotools, Hamburg, Germany), actin and FLAG-tag (Sigma, St. Louis, MO), Src (Upstate, Waltham, MA), and Phospho-Tyr529-Src and phospho-tyr418-Src (Invitrogen).

      DNA Constructs

      The EGFR coding sequence was amplified from the pBSII-SK-EGFR plasmid kindly provided by Dr. William Pao (MSKCC, New York, NY). EGFR coding sequences were inserted into pTre2hyg (Clontech, Palo Alto, CA) using the NheI and NotI cloning sites to produce pTre2hyg-EGFR-Δ746-750-FLAG (control) or pTre2hyg-EGFR-Δ746-750-T790M-FLAG. To insert the NheI site at the 5′ of the EGFR coding sequence, the following forward primer was used: 5′-GCA ATG GCT AGC ACC ATG CGA CCC TCC GGG ACG-3′. A FLAG-tag (DYKDHD) followed by a NotI site were inserted at the 3′ end of the EGFR coding sequence using the following primer: 5′-GCA ATG GCG GCC GCC TAC TTG TCA TCG TCA TCC TTG TAA TCA CCT GCT CCA ATA AAT TC 3′. Site-directed mutagenesis was performed by polymerase chain reaction using the following primers: 5′-GTC GCT ATC AAG ACA TCT CCG AAA GCC-3′ (in-frame deletion of amino acids 746–750) and 5′ GTG CAG CTC ATC ATG CAG CTC ATG CCC 3′ (T790M mutation). Polymerase chain reaction products were fully sequenced after ligation into pTre2hyg vector. The Module 1 plasmid (pN1pβactin-rtTA2S-M2-IRES-EGFP)
      • Welman A
      • Barraclough J
      • Dive C
      Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2.
      confers constitutive polycistronic expression of the tetracycline reverse transcriptional transactivator rtTA 2 S-M 2 (“tet-on”) and the eGFP gene and resistance to G418.

      Cell Culture and Production of Stable Cell Lines

      HCC827 cells were from DSMZ (Braunschweig, Germany). H3255 cells were a gift from Dr. Ming-Sound Tsao (OCI, Toronto, Canada). All cells were maintained in RPMI medium with 10% fetal bovine serum (FBS), which was not supplemented with insulin-like growth factor in any experiments. All experiments were performed in the presence of FBS unless otherwise stated. For stable cell line generation, HCC827 cells were electroporated with the Module 1 plasmid and selected with 600 μg/ml G418. Cells positive for eGFP (i.e., also expressing rtTA2S-M2) were selected by fluorescence activated cell sorting. These “HCC827-M1” cells were subsequently transfected with pTre2hyg-EGFR-Δ746-750-FLAG (control) or pTre2hyg-EGFR-Δ746-750-T790M-FLAG and selected with 50 μg/ml hygromycin and 600 μg/ml G418. Inducible expression of EGFR Δ746-750 in HCC827 cells (which already express this protein) was used as a control to evaluate the effect of increased exogenous expression of the deletion mutation alone when compared with the deletion mutation with added T790M (del746-750/T790M).
      Stably transfected cell populations were cloned by low density seeding in a 1:1 mixture of conditioned and fresh media containing G418 and hygromycin as above. Clones capable of inducible expression of EGFR mutants were identified by Western blotting for EGFR-FLAG expression.

      Western Blotting

      Cells were prepared for Western blotting by direct lysis in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer (62.5 mmol/L Tris [pH 6.8], 2% SDS, 10% glycerol, and 5% b-mercaptoethanol) or by extraction in cell lysis buffer (cell signaling) followed by protein quantification using the bicinchoninic acid assay (Pierce) and lysis in Laemmli sample buffer. Protein samples were run into SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes. Membranes were probed with primary antibodies overnight at 4°C and with secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature. Blots were developed by enhanced chemiluminescence and photographed using a Fujifilm Dark Box II and the Image Reader LAS-1000 Plus software.

      3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay

      Cells were tested for sensitivity to different compounds using the MTT assay. Briefly, cells were seeded onto 96-well plates at a density of 2.5 × 103 cells per well and allowed to attach for 24 hours at 37°C before drug treatment. After 72 hours of drug exposure, the medium was removed and replaced with 50 μL of 3 mg/ml MTT (Sigma) in phosphate buffered saline. Cells were incubated in the MTT solution for 4 hours at 37°C, after which it was removed, and 100 μL DMSO were added per well to solubilize the formazan product. After solubilization, 25 μL of Tris-HCl pH 10.5 were added per well, and the plates were read in a Multiskan EX plate reader with λem 540 nm. Data were analyzed using the GraphPad Prism software.

      Cell Proliferation Analysis

      Cells were plated into six-well plates at 1 × 105 cells/well and allowed to attach for 24 hours. Thereafter, medium was changed for: RPMI supplemented with FBS, with or without 100 ng/ml of doxycycline; or FBS-free RPMI (serum starvation), with or without 100 ng/ml of doxycycline. Before serum starvation, cells were washed twice in FBS-free RPMI. Cell counts were performed in an ADAM cell counter (Digital Bio) every 24 hours for 96 hours after addition of medium ± doxycycline. Medium was changed at 48 hours to prevent growth arrest. Data were analyzed using the GraphPad Prism software.

      Transmission Electron Microscopy (TEM)

      Drug-treated cells plated on 10 cm2 cell culture dishes were fixed with 2% EM Grade Glutaraldehyde in 0.2 M HEPES buffer, pH 7.4 for 2 hours at room temperature. Cells were scraped, transferred to tubes, and postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 hour and then with 1% uranyl acetate for 1 hour in the dark. Dehydration in a series of 50 to 100% ethanol and 100% propylene oxide was followed by embedding in TAAB LV resin. Ultrathin sections cut using a Reichert Ultracut S ultramicrotome were stained with lead citrate before observation in a FEI Tecnai 12 Bio Twin transmission electron microscope at 80 kV.

      Light Microscopy

      The acidophilic dye Lysotracker green (Molecular Probes) was used to identify acidic vesicles in drug-treated cells. HCC827 cells were plated onto glass coverslips and drug-treated with 3 μM PI103 for 16 hours at 37°C. Lysotracker green (50 nM) was added 15 hours after treatment. Real-time imaging of cells in medium without Lysotracker green was performed using a Zeiss Axiovert 200 M light microscope equipped with a Roper Cascade 512B cooled charge coupled device camera. Images were analyzed using the software Metamorph.

      Flow Cytometry

      HCC827 or H3255 cells were treated with different concentrations of PI103 for 16 hours before quantitation of the cellular accumulation of acidic vesicles. After 15 hours of treatment, fresh medium was provided containing Lysotracker green (100 nM) and the appropriate concentration of PI103. At the end of the treatment period, cells were trypsinized, spun, and resuspended in 400 μL of phosphate buffered saline before analysis in a BD FACSCalibur flow cytometer (λex 488 nm, λem 530 ± 30 nm). Flow cytometric data were analyzed using the FlowJo software (BD).

      RESULTS

      HCC827 Clones Inducibly Expressing EGFR-Del/T790M Were Resistant to Gefitinib and Less Sensitive to CL-387785

      HCC827 NSCLC cells express an activated EGFR mutant (exon 19 in-frame deletion of amino acids 746–750, hereafter referred to as EGFR-Del). These cells are sensitive to gefitinib and erlotinib and were derived from a lung adenocarcinoma of a nonsmoking woman.
      • Girard L
      • Zochbauer-Muller S
      • Virmani AK
      • et al.
      Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering.
      • Amann J
      • Kalyankrishna S
      • Massion PP
      • et al.
      Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer.
      Therefore, HCC827 cells exhibit the clinicopathologic characteristics of NSCLC sensitive to EGFR TKIs.
      HCC827 clones capable of inducible expression of EGFR mutants were generated by two steps of transformation and selection, and tested for inducible expression of FLAG-tagged EGFR-Del or EGFR-Del/T790M by Western blotting. Two representative clones of each type were selected with the lowest expression of FLAG-EGFR in the absence of doxycycline (Figure 1A). These clones also showed homogeneous expression of the FLAG-tagged protein by immunofluorescence (Figure S1). For simplicity, the clones chosen to be used in further studies were renamed D1 and D2 (for EGFR-Del-FLAG clones 1 and 2) and T1 and T2 (for EGFR-Del/T790M-FLAG clones 1 and 2). Parental cells did not express FLAG, and none of the protein levels were altered by the presence or absence of doxycycline. All clones showed a clear increase in total levels of EGFR protein after treatment with doxycycline, which was accompanied by increased levels of phosphorylated EGFR (Figures 1A and S2). Densitometric analysis on the Western blots shown in Figure S2 indicated that induction of EGFR-Del/T790M (with 100 ng/ml doxycycline) resulted in no more than a twofold increase in the expression of EGFR. Treatment of parental cells with gefitinib or CL-387785 caused marked inhibition of p-EGFR, p-Akt, and p-ERK but did not affect the levels of any other proteins, consistent with inhibition of EGFR signaling pathway. EGFR, ERK 1 and 2, and Akt remained active (as determined by phosphorylation) after treatment of clones T1 and T2 (but not D1 and D2) with 1 μM gefitinib in the presence of doxycycline. In contrast, EGFR pathway inactivation (measured by dephosphorylation of EGFR, Akt, and ERK1/2) occurred in all clones after treatment with 1 μM of the EGFR irreversible inhibitor CL-387785 (Figure 1A).
      Figure thumbnail gr1
      FIGURE 1Inducible expression of EGFR T790M in HCC827 cells keeps the EGFR pathway active after gefitinib treatment, resulting in resistance to this drug. A, D and T clones were grown in the presence or absence of doxycycline for 24 hours, treated with 1 μM gefitinib or CL-387785 for 3 hours, and prepared for Western blotting for detection of the FLAG-tagged EGFR, EGFR, ERK1/2, and Akt. The activated forms of these proteins—phospho-Tyr1068-EGFR, phospho-Ser473-Akt, and phospho-Thr202/Tyr204–ERK1/2—are represented as P-EGFR, P-Akt, and P-ERK1/2, respectively. Actin was used as a loading control. B, D and T clones were treated with increasing concentrations of gefitinib or CL-387785 for 72 hours, in the presence or absence of doxycycline, and then subjected to MTT assays for drug sensitivity. Data in the y axis were represented as the % Abs. relative to that of the DMSO-treated control. EGFR, epidermal growth factor receptor; DOX, doxycycline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide.
      Parental HCC827 cells were sensitive to gefitinib in MTT assays measured 72 hours after drug exposure (Figure 1B). Clones D1 and D2 showed similar sensitivity to gefitinib as parental cells, even after induced EGFR-Del expression. In contrast, clones T1 and T2 showed marked doxycycline-dependent resistance to gefitinib (IC50 >5 μM) and decreased sensitivity to CL-387785 (Figure 1B). A small decrease in gefitinib sensitivity was observed in T clones without doxycycline, probably due to residual EGFR-Del/T790M expression in these conditions. Induced EGFR-Del/T790M-FLAG expression, gefitinib resistance, and decreased sensitivity to CL-387785 were doxycycline concentration-dependent and reached maximal levels after treatment with 12.5 to 25 ng/ml of doxycycline (Figure S2).

      Induced EGFR-Del/T790M Expression Did Not Provide a Clear Cell Proliferation Advantage to HCC827 Cells in Culture

      The isogenic, clinically relevant HCC827 cell model described here is ideally suited to identify changes in proliferation and signaling conferred by EGFR T790M. Selected clones were grown in medium containing 10% FBS or in serum-free medium for 96 hours, in the presence or absence of doxycycline, to determine whether EGFR-Del/T790M induction conferred a proliferation advantage. No significant changes in proliferation were seen in any of the clones tested and parental cells, although slight proliferation changes could be attributed to clonal variation (Figure 2A). Proliferation indexes are shown in Table S1.
      Figure thumbnail gr2
      FIGURE 2Cell proliferation and the EGFR phosphorylation pattern were not affected by inducible expression of EGFR T790M in HCC827 cells. A, Cell proliferation of D and T clones was monitored for 96 hours in the presence or absence of FBS and/or doxycycline in the culture medium. Fresh medium was added at 48 hours to prevent cell cycle arrest. B, Clones were grown for 24 hours in the presence or absence of doxycycline and treated with gefitinib for 3 hours before sample preparation for Western blotting for total or phosphorylated EGFR. Note that the amino acid numbering used to describe phosphorylated tyrosine residues in EGFR disregards its 24-amino acid long N-terminal signal sequence. EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; DOX, doxycycline; DMSO, dimethyl sulfoxide.
      In agreement with the cell proliferation analysis, no obvious increase in ERK1/2 or Akt phosphorylation was observed when T clones were treated with doxycycline (Figure 1A), despite the fact that EGFR phosphorylation at seven different tyrosine residues was increased in all clones in these conditions (Figure 2B). Phosphorylation at tyrosine 1045 of EGFR is required for down-regulation of EGFR signaling by internalization and degradation, and low levels of phosphorylation at this site probably contribute to the potency of signaling of activated EGFR mutants.
      • Shtiegman K
      • Kochupurakkal BS
      • Zwang Y
      • et al.
      Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling.
      After treatment with doxycycline, T clones showed a greater increase in phosphorylation of tyrosine 1045 than D clones (Figure 2B).
      To our knowledge, the only report of differential signaling activation resulting from the addition of T790M mutation to activated EGFR was the increased activation of c-Src in NIH3T3 cells expressing EGFR double mutants containing the T790M mutation.
      • Godin-Heymann N
      • Bryant I
      • Rivera MN
      • et al.
      Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
      These cells also became rounded and less substrate adherent when compared with those expressing single (activated) mutants.
      • Godin-Heymann N
      • Bryant I
      • Rivera MN
      • et al.
      Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
      These changes were not confirmed in this study: activating phosphorylation of c-Src at the tyrosine residue 418 was not detected in D or T clones grown in the presence or absence of doxycycline (Figure S3). In contrast, the inhibitory phosphorylation of tyrosine 529 in c-Src was readily detected in all clones and conditions (Figure S3). This suggests that c-Src is inactive in the clinically relevant HCC827 cells (in routine culture conditions), even after inducible expression of EGFR T790M. In agreement with this, no morphologic transformation was observed after treatment of D or T clones with doxycycline (not shown).

      HCC827 Parental Cells and Clones Were Not Sensitive to MEK Inhibition But Were Sensitive to Class I PI3K Inhibition by PI103

      A logical approach to overcome EGFR T790M-mediated drug resistance is to target downstream signaling molecules such as MEK, PI3K, or mTOR. In this study, two MEK inhibitors (U0126 and PD98059) and one PI3K/mTOR inhibitor (PI103) were evaluated in parental HCC827 cells and in T and D clones, in the presence or absence of doxycycline. Importantly, although PI103 inhibits class I PI3Ks preferentially,
      • Raynaud FI
      • Eccles S
      • Clarke PA
      • et al.
      Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.
      it most likely also inhibits mTOR (both mTORC1 and mTORC2 complexes) at the highest concentrations used here.
      • Fan QW
      • Knight ZA
      • Goldenberg DD
      • et al.
      A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.
      HCC827 parental and clonal cell lines were resistant to MEK inhibitors at concentrations that span the IC50 of most cancer cell lines
      • Favata MF
      • Horiuchi KY
      • Manos EJ
      • et al.
      Identification of a novel inhibitor of mitogen-activated protein kinase kinase.
      (Figure 3A and Table 1). In contrast, these cells were sensitive to PI103, with IC50 values in the range of 0.3 to 0.8 μM (Figure 3A and Table 1). A different EGFR TKI-sensitive cell line (H3255, which expresses EGFR L858R) was treated with U0126 or with PI103 to verify if sensitivity to PI3K inhibition was unique to HCC827 cells or was found in other NSCLC cells dependent on an activating EGFR mutant. Although H3255 cells were less resistant to MEK inhibition than HCC827 (IC50 = 7.84 μM for U0126), their sensitivity to PI103 was in the same range as for HCC827 cells (Table 1).
      Figure thumbnail gr3
      FIGURE 3HCC827 parental cells and clones are sensitive to PI3K/mTOR inhibition by PI103 but are resistant to MEK inhibition. A, Cells were treated with increasing concentrations of U0126 or PI103 for 72 hours, in the presence or absence of doxycycline, and subjected to the MTT assay. Data in the y axis were represented as the % absorbance relative to that of the DMSO-treated control. B and C, HCC827 or H3255 cells were treated with PI103, U0126, or PD98059 for 16 hours and prepared for Western blotting for the detection of total and phosphorylated ERK1/2, Akt, p70 S6K, or total BIM (only the bands corresponding to the BIMEL isoform are shown). Actin was used as a loading control. In B, drug concentrations are displayed parenthetically. MEK, extracellular signal-regulated kinase kinase; mTOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ERK, extracellular signal-regulated kinase; PI, PI103; U0, U0126; PD, PD98059.
      TABLE 1Sensitivity of NSCLC Cell Lines to MEK and PI3K/mTOR Inhibitor
      Cell LineDoxycyclineIC50 Values (μM)
       PI-103U0126PD98059GefitinibCL-387,785
       HCC8270.74>10>200.0150.001
       HCC827+0.62>10>200.0190.001
       Clone D10.88>10>200.0210.005
       Clone D1+0.76>10>200.0240.005
       Clone D20.58>10>200.0050.004
       Clone D2+0.46>10>200.0050.002
       Clone T10.75>10>200.1830.114
       Clone T1+0.46>10>20>50.901
       Clone T20.59>10>200.1250.092
       Clone T2+0.40>10>20>51.005
       H32550.497.84n.d.0.0210.001
      Parental and clonal cell lines were subjected to MTT assays after 72 h of drug exposure, in the presence or absence of doxycycline. IC50 values were calculated using the software GraphPad Prism.
      NSCLC, non-small cell lung cancer; MEK, extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; n.d., not done.
      Phosphorylation of ERK1/2 after treatment with PD98059 and U0126 was examined as a proof of mechanism and pharmacodynamic biomarker of the activity of the MEK inhibitors used. BIMEL phosphorylation and protein level were also examined before and after treatment with MEK inhibitors. Treatment with 10 or 20 μM of U0126 for 20 hours resulted in a clear decrease in ERK phosphorylation, an increase in BIMEL protein levels, and a clear mobility shift in this protein compatible with dephosphorylation (Figure 3B). UO126 at concentrations of 1.25, 2.5, and 5 μM did not provoke a decrease in ERK phosphorylation in HCC827 cells. These data (Figure S4) together with that presented in Figure 3B show that whether ERK was phosphorylated (UO126 <10 μM) or not (UO126 10 μM or 20 μM), there was no effect on cell population growth. Therefore, at the highest concentration of U0126 used in the MTT assay (10 μM), apoptosis-associated changes in BIMEL identical to those reported in the literature
      • Cragg MS
      • Kuroda J
      • Puthalakath H
      • et al.
      Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
      • Gong Y
      • Somwar R
      • Politi K
      • et al.
      Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas.
      were observed, despite the fact that cells were insensitive to MEK inhibition (Figure 3A). PD98059 was not as effective as U0126 in decreasing ERK phosphorylation and modulating BIMEL, consistent with its reduced potency compared with U0126.
      • Favata MF
      • Horiuchi KY
      • Manos EJ
      • et al.
      Identification of a novel inhibitor of mitogen-activated protein kinase kinase.
      HCC827 and H3255 cells were considerably more sensitive to PI3K/mTOR inhibition than to MEK inhibition (Figure 3A and Table 1). Lower concentrations of PI103 were needed to reduce phosphorylation of Akt and p70S6K (a biomarker of mTOR activity) in HCC827 compared with H3255 (Figure 3C), despite the fact that these cell lines were similarly sensitive to PI103. Activating phosphorylation of Akt or p70S6K was undetectable in all cell lines after treatment with 3 μM PI103 (Figure 3B).
      UO126 when applied as a single agent at 10 μM had little or no effect on HCC827 (parental and induced clones) cell growth, despite clear inhibition of p-ERK (Figure 3B)). The effect of combining UO126 (10 μM) with PI-103 was investigated in parental and induced and uninduced clones (D1, D2, T1, and T2). There was no significant effect of combining this PI3K inhibitor with this MEK inhibitor in these cells (Figure S5).

      PI103 Promoted Autophagy in HCC827 and H3255 Cells

      Recently, PI103 treatment of two cancer cell lines was shown to induce autophagy.
      • Degtyarev M
      • De Maziere A
      • Orr C
      • et al.
      Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
      A hallmark of autophagy is cytoplasmic accumulation of acidic vacuoles resulting from fusion of early autophagic vacuoles (autophagosomes) to lysosomes or other acidic compartments from the endocytic pathway.
      • Klionsky DJ
      • Abeliovich H
      • Agostinis P
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
      Treatment of HCC827 or H3255 cells with 3 μM PI103 resulted in accumulation of cytoplasmic acid vacuoles identified by labeling with Lysotracker green (Figure 4A). Cell populations treated with 3 μM PI103 displayed increased intensity of Lysotracker green fluorescence (Figure 4B) allowing for the quantification of acidic vesicle accumulation by flow cytometry (Figure 4C). In HCC827 cells, a progressive increase in accumulation of acidic vesicles was observed after treatment with 0.125 to 3 μM of PI103 (Figure 4C). In H3255 cells, acidic vesicle accumulation was evident only after treatment with 1 or 3 μM of PI103 (Figure 4C).
      Figure thumbnail gr4
      FIGURE 4Autophagy was induced by treatment of EGFR-TKI sensitive cell lines with PI103. A, HCC827 cells were labeled with Lysotracker green during the last hour of treatment with DMSO or PI103 and observed live in an inverted microscope at a magnification of 40X. B and C, Flow cytometry analysis of cells treated with PI103 or DMSO and labeled with Lysotracker green. B, Cells treated with 3 μM of PI103 show a clear increase in Lysotracker green fluorescence, indicating the accumulation of acidic vacuoles. The R1 gate shown in this graph was arbitrarily defined so as to include cells with increased Lysotracker green fluorescence compared with control, DMSO-treated cells (i.e., this gate excluded >90% of control cells). C, Quantification of cells found in the R1 gate after treatment with increasing concentrations of PI103 for 16 hours. D, HCC827 and H3255 cells were treated with different compound for 16 hours and prepared for Western blotting for the detection of the autophagy marker LC3-II or the apoptosis marker cleaved PARP. Actin was used as a loading control. The numbers in parenthesis after compound names represent the concentrations in μM. E, D or T clones were grown in the presence or absence of doxycycline for 24 hours before 16 hour-treatment with DMSO or PI103 and Western blotting for the detection of LC3. F, HCC827 cells were treated for 16 hours with DMSO or PI103 alone or in combination with the lysosomal protease inhibitors E64d and Pepstatin A before Western blotting for LC3 detection. EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; DMSO, dimethyl sulfoxide; PI, PI103; U0, U0126; PD, PD98059; Gef., gefitinib; CL, CL-387785; Rap., rapamycin.
      Because PI103 treatment resulted in accumulation of cytoplasmic acidic vesicles suggestive of autophagy, an established autophagy biomarker was sought. During autophagy, the protein LC3-I is incorporated into membrane of autophagosomes via coupling to phosphatidylethanolamine.
      • Kabeya Y
      • Mizushima N
      • Ueno T
      • et al.
      LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
      This lipidated form of LC3-I is termed LC3-II. The electrophoretic mobility shift associated with conversion of LC3-I to LC3-II during autophagosome formation is detectable on LC3 western blots.
      • Klionsky DJ
      • Abeliovich H
      • Agostinis P
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
      • Kabeya Y
      • Mizushima N
      • Ueno T
      • et al.
      LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
      A demonstrable increase in LC3-II, accompanied by a decrease in LC3-I, was observed when HCC827 (parental cells and induced and noninduced clones) or H3255 cells were treated with 1 or 3 μM of PI103 for 16 hours (Figures 4D and E). Treatment of HCC827 (induced and noninduced clones) with PI103 (1 or 3 μM, 16 hours) did not result in cleavage of PARP (Figure 4E). LC3-II is degraded late in autophagy, when autophagosomes fuse with lysosomes, forming autolysosomes. Therefore, increases in LC3-II levels might indicate autophagy induction or a block in protein degradation at the autolysosome.
      • Mizushima N
      • Yoshimori T
      How to interpret LC3 immunoblotting.
      To determine whether PI103 induced or blocked autophagy, HCC827 cells were treated with PI103 in the presence of the lysosomal protease inhibitors E64d and Pepstatin A. No increase in LC3-II levels in these conditions indicates a block in autophagy.
      • Mizushima N
      • Yoshimori T
      How to interpret LC3 immunoblotting.
      However, treatment with lysosomal protease inhibitors and PI103 generated further accumulation of LC3-II when compared with PI103 treatment alone (Figure 4F), showing that PI103 induced autophagy.
      Little or no cleaved PARP (a biomarker of apoptosis) was detected in HCC827 or H3255 cells treated with PI103 for 16 hours (Figure 4D). Treatment with gefitinib or CL-387785 was used as a positive control for the detection of apoptosis. Surprisingly, these EGFR TKIs did not elicit apoptosis in HCC827 cells after 16 hours of treatment, whereas a strong signal corresponding to cleaved PARP was observed readily after treatment of H3255 cells in the same conditions (Figure 4D). Close examination of data in the literature revealed that induction of apoptosis by gefitinib or erlotinib seems to be delayed in HCC827 compared with H3255 cells.
      • Costa DB
      • Halmos B
      • Kumar A
      • et al.
      BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations.
      • Amann J
      • Kalyankrishna S
      • Massion PP
      • et al.
      Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer.
      Treatment of cells with PI-103 or gefitinib did not result in apoptosis, as assessed by cleaved PARP in HCC827 cells, up to 48 hours of drug exposure (Figure S6). Indeed, a recent report described the effect of gefitinib as mainly cytostatic in a NSCLC cell line expressing EGFR-Del (PC-9), in contrast to the proapoptotic effect observed with H3255 cells.
      • Pratilas CA
      • Hanrahan AJ
      • Halilovic E
      • et al.
      Genetic predictors of MEK dependence in non-small cell lung cancer.
      Treatment with the autophagy inducer rapamycin (an mTORC1 inhibitor) increased LC3-II levels in H3255 cells (but not in HCC827) (Figure 4D).
      Autophagy induction by PI103 was confirmed ultrastructurally by the observation of autophagic vacuoles in thin sections of TEM. HCC827 cells treated with DMSO displayed normal cytoplasmic morphology in thin-section TEM, with no evidence of increased autophagy (Figure 5A). In contrast, different types of autophagic vacuoles, including autophagosomes (with double membrane and cytoplasmic content) and smaller or larger (multivesicular) autolysosomes (i.e., late autophagic vacuoles) were often found by thin-section TEM in HCC827 cells treated with PI103 (Figures 5BD), confirming PI103 induced autophagy.
      Figure thumbnail gr5
      FIGURE 5Autophagy induction by PI103 was confirmed by ultrastructural observation of treated cells by TEM. A, Control, DMSO-treated cells did not display signs of increased autophagy. B, C, and D, Early (arrowheads) or late (arrows) autophagic vacuoles (autophagosomes or autolysosomes, respectively) could be found in the cytoplasm of cells treated with PI103 for 16 hours. TEM, transmission electron microscopy; DMSO, dimethyl sulfoxide; N, nucleus.

      DISCUSSION

      The discovery of a link between activating EGFR mutations in clinical NSCLC and response to the EGFR TKIs gefitinib and erlotinib
      • Lynch TJ
      • Bell DW
      • Sordella R
      • et al.
      Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.
      • Paez JG
      • Janne PA
      • Lee JC
      • et al.
      EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.
      • Pao W
      • Miller V
      • Zakowski M
      • et al.
      EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib.
      was a milestone in the field of personalized cancer therapy. However, resistance emerges in all patients who initially responded to these agents highlighting the importance of seeking novel therapies for lung cancers dependent on mutant EGFR activity. Knowledge of the cell biology and drug sensitivity of cells expressing drug resistance mutations must be considered in the design of novel agents to prevent or overcome acquired drug resistance in the clinic. The inducible expression of EGFR T790M in HCC827 cells described here represents a robust, isogenic system to study the properties conferred by this important drug resistance mutant to a clinically relevant cell line.
      It is not surprising that inducible expression of EGFR-Del/T790M in HCC827 cells did not enhance cell proliferation, increase activity of Akt, ERK1/2 or c-Src, or result in obvious alterations of cell morphology, despite the fact that all clones displayed increased EGFR phosphorylation after doxycycline treatment. The T790M mutation in cis with an activating EGFR mutation has been detected as a minor clone in clinical NSCLC samples before treatment with gefitinib or erlotinib,
      • Inukai M
      • Toyooka S
      • Ito S
      • et al.
      Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer.
      • Tokumo M
      • Toyooka S
      • Ichihara S
      • et al.
      Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer.
      • Maheswaran S
      • Sequist LV
      • Nagrath S
      • et al.
      Detection of mutations in EGFR in circulating lung-cancer cells.
      • Sequist LV
      • Martins RG
      • Spigel D
      • et al.
      First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations.
      suggesting that these cells do not display improved proliferation in vivo. Rather, EGFR T790M expressing cells seem to be selectively amplified during drug treatment.
      To date, studies associating EGFR T790M expression (in a background of an activating EGFR mutation) to altered signaling or growth properties were performed by constitutive expression and/or in cell systems where sensitivity to EGFR TKIs was generated artificially.
      • Godin-Heymann N
      • Bryant I
      • Rivera MN
      • et al.
      Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
      • Yun CH
      • Mengwasser KE
      • Toms AV
      • et al.
      The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.
      • Engelman JA
      • Mukohara T
      • Zejnullahu K
      • et al.
      Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer.
      These systems are not isogenic (when constitutive expression is established) and/or might not be representative of the context of NSCLC sensitive to EGFR TKIs, due to the choice of cell line. Our results suggest that, in EGFR TKI-sensitive NSCLC cells, where EGFR activity is inherently increased and required for proliferation and survival, further activation of downstream signaling pathways by increased mutant EGFR expression is prevented by regulatory mechanisms. Mulloy et al.
      • Mulloy R
      • Ferrand A
      • Kim Y
      • et al.
      Epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib.
      report that the EGFR T790M/L858R double mutant showed increased oncogenic properties in comparison with the single mutants. This was in contrast to the data presented here. This may be explained by the different cell models employed to study the effect of the T790M mutation. This highlights the importance of studying mutant EGFR activity by inducible (rather than constitutive) expression in a clinically relevant cell background.
      The in vitro data presented here does not, however, exclude the possibility that changes mediated by acquired EGFR T790M mutation in vivo have subtle effects on tumor biology or on tumorigenesis, which might underlie the observation that, in a mouse model of EGFR L858R/T790M-induced lung tumorigenesis, EGFR L858R/T790M expression resulted in formation of bronchial papillary adenocarcinomas as well as the peripheral adenocarcinomas generated by EGFR L858R expression alone.
      • Li D
      • Shimamura T
      • Ji H
      • et al.
      Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.
      Inducible EGFR-Del/T790M expression in HCC827 cells faithfully recapitulated the resistance to lower concentrations of irreversible EGFR inhibitors observed in model systems, and reinforced the idea (supported by early clinical data) that these agents have modest activity against NSCLC with acquired resistance to gefitinib and erlotinib.
      • Li D
      • Shimamura T
      • Ji H
      • et al.
      Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.
      • Godin-Heymann N
      • Ulkus L
      • Brannigan BW
      • et al.
      The T790M “gatekeeper” mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor.
      • Wong KK
      HKI-272 in non small cell lung cancer.
      MEK inhibition alone is also unlikely to sensitize this type of NSCLC, according to our data and that of others.
      • Pratilas CA
      • Hanrahan AJ
      • Halilovic E
      • et al.
      Genetic predictors of MEK dependence in non-small cell lung cancer.
      Interestingly, our data show that up-regulation and dephosphorylation of BIMEL alone, which occur on MEK inhibition, are insufficient stimulus for apoptosis in NSCLC cells with activating EGFR mutations. Treatment with EGFR TKIs must elicit additional, MEK-independent, proapoptotic stimuli to drive apoptosis in this context. It remains possible that the combination of MEK inhibitors with agents providing alternative proapoptotic stimuli will be effective against EGFR TKI-resistant NSCLC.
      Importantly, our data show that NSCLC cells with acquired resistance to gefitinib and erlotinib remain sensitive to PI3K/mTOR inhibition. Treatment of HCC827 or H3255 cells with PI103 resulted in markedly increased autophagy, but little or no apoptosis. PI103 induces autophagy in PC3 and U87MG cancer cell lines,
      • Degtyarev M
      • De Maziere A
      • Orr C
      • et al.
      Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
      although the relationship between autophagy induction and Akt/mTOR signaling down-regulation was not examined. In this study, autophagy induction after treatment with 3 μM of PI103 was associated with the lowest levels of Akt/mTOR activity, as determined by phosphorylation of Akt and p70S6K. At this concentration, PI103 is expected to inhibit class I PI3K and both mTOR containing complexes mTORC1 and mTORC2,
      • Raynaud FI
      • Eccles S
      • Clarke PA
      • et al.
      Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.
      and this might be required for overt autophagy induction.
      The relationship between induction of a moderate degree of autophagy and Akt/mTOR signaling down-regulation was complex and dependent on cell context. In HCC827 cells, decreased phosphorylation of Akt and p70S6K occurred at lower concentrations of PI103 (<1 μM) than those required for autophagy induction according to LC3-II accumulation. However, some accumulation of acidic vesicles occurred after treatment with 0.125 to 0.5 μM PI103. Subtle increases in the transient autophagy marker LC3-II (suggestive of some autophagy induction) might have occurred after treatment with lower concentrations of PI103 but were undetectable with the assays used. In H3255 cells, autophagy was induced by 1 μM of PI103 without obvious net down-regulation of Akt or mTOR signaling. The moderate degree of autophagy induced by 1 μM PI103 in H3255 cells might result from the inhibition of mTOR-independent outputs of Akt activity, for which Ser 473 dephosphorylation is not a reliable biomarker. In fact, inhibition of Akt kinase activity results in increased (rather than decreased) phosphorylation of Akt on Ser 473, despite the fact that mTOR-independent outputs of Akt activity are clearly inhibited.
      • Rhodes N
      • Heerding DA
      • Duckett DR
      • et al.
      Characterization of an Akt kinase inhibitor with potent pharmacodynamic and antitumor activity.
      Taken together, the data presented here suggest that combined inhibition of PI3K and mTOR could be of clinical relevance in treatment of NSCLC with activating EGFR mutations and for the prevention/treatment of acquired resistance to EGFR TKIs. Our data also highlight the fact that robust PI3K and mTOR inhibition are likely to provoke autophagy. Although PI3K/mTOR inhibitors might provide patient benefit due to a block of tumor cell proliferation, the concomitant induction of autophagy might have a more lasting negative effect of increasing tumor cell survival. Comparison of the data presented in this work with that of others
      • Fan QW
      • Knight ZA
      • Goldenberg DD
      • et al.
      A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.
      • Raynaud FI
      • Eccles S
      • Clarke PA
      • et al.
      Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.
      • Fan QW
      • Cheng CK
      • Nicolaides TP
      • et al.
      A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma.
      suggests that robust autophagy induction occurs at drug concentrations higher than those required for cell proliferation inhibition. Therefore, it is possible that a therapeutic window for PI3K/mTOR inhibitors could be identified where cell proliferation is inhibited, with minimal autophagy induction. However, further studies are now necessary to determine the potential role of autophagy in this context as a cell death rather than survival mechanism and the potential to switch autophagic cell to apoptosis.

      ACKNOWLEDGMENTS

      The authors thank Dr. Donald Ogilvie (Head of Drug Discovery, Paterson Institute for Cancer Research, UK) for constructive critical review of this article and invaluable comments and discussion. The authors thank Dr Arek Welman (Beatson Institute for Cancer Research, Glasgow, Scotland) for advice regarding the doxycycline inducible cell system. Flavia F. Moreira-Leite was funded by AstraZeneca. The Paterson Institute for Cancer Research is core-funded by Cancer Research UK.

      REFERENCES

        • Yarden Y
        • Sliwkowski MX
        Untangling the ErbB signalling network.
        Nat Rev Mol Cell Biol. 2001; 2: 127-137
        • Lynch TJ
        • Bell DW
        • Sordella R
        • et al.
        Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.
        N Engl J Med. 2004; 350: 2129-2139
        • Paez JG
        • Janne PA
        • Lee JC
        • et al.
        EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.
        Science. 2004; 304: 1497-1500
        • Pao W
        • Miller V
        • Zakowski M
        • et al.
        EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib.
        Proc Natl Acad Sci USA. 2004; 101: 13306-13311
        • Sharma SV
        • Bell DW
        • Settleman J
        • et al.
        Epidermal growth factor receptor mutations in lung cancer.
        Nat Rev Cancer. 2007; 7: 169-181
        • Engelman JA
        • Janne PA
        Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.
        Clin Cancer Res. 2008; 14: 2895-2899
        • Kobayashi S
        • Boggon TJ
        • Dayaram T
        • et al.
        EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.
        N Engl J Med. 2005; 352: 786-792
        • Pao W
        • Miller VA
        • Politi KA
        • et al.
        Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.
        PLoS Med. 2005; 2: e73
        • Bean J
        • Brennan C
        • Shih JY
        • et al.
        MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.
        Proc Natl Acad Sci USA. 2007; 104: 20932-20937
        • Engelman JA
        • Zejnullahu K
        • Mitsudomi T
        • et al.
        MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.
        Science. 2007; 316: 1039-1043
        • Balak MN
        • Gong Y
        • Riely GJ
        • et al.
        Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.
        Clin Cancer Res. 2006; 12: 6494-6501
        • Costa DB
        • Halmos B
        • Kumar A
        • et al.
        BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations.
        PL oS Med. 2007; 4 (discussion 1680.): 1669-1679
        • Bean J
        • Riely GJ
        • Balak M
        • et al.
        Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma.
        Clin Cancer Res. 2008; 14: 7519-7525
        • Blencke S
        • Zech B
        • Engkvist O
        • et al.
        Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors.
        Chem Biol. 2004; 11: 691-701
        • Regales L
        • Balak MN
        • Gong Y
        • et al.
        Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.
        PLoS One. 2007; 2: e810
        • Godin-Heymann N
        • Bryant I
        • Rivera MN
        • et al.
        Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.
        Cancer Res. 2007; 67: 7319-7326
        • Li D
        • Shimamura T
        • Ji H
        • et al.
        Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.
        Cancer Cell. 2007; 12: 81-93
        • Vikis H
        • Sato M
        • James M
        • et al.
        EGFR-T790M is a rare lung cancer susceptibility allele with enhanced kinase activity.
        Cancer Res. 2007; 67: 4665-4670
        • Yun CH
        • Mengwasser KE
        • Toms AV
        • et al.
        The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.
        Proc Natl Acad Sci USA. 2008; 105: 2070-2075
        • Bell DW
        • Gore I
        • Okimoto RA
        • et al.
        Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR.
        Nat Genet. 2005; 37: 1315-1316
        • Engelman JA
        • Mukohara T
        • Zejnullahu K
        • et al.
        Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer.
        J Clin Invest. 2006; 116: 2695-2706
        • Mulloy R
        • Ferrand A
        • Kim Y
        • et al.
        Epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib.
        Cancer Res. 2007; 67: 2325-2330
        • Shimamura T
        • Li D
        • Ji H
        • et al.
        Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance.
        Cancer Res. 2008; 68: 5827-5838
        • Godin-Heymann N
        • Ulkus L
        • Brannigan BW
        • et al.
        The T790M “gatekeeper” mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor.
        Mol Cancer Ther. 2008; 7: 874-879
        • Wong KK
        HKI-272 in non small cell lung cancer.
        Clin Cancer Res. 2007; 13: s4593-s4596
        • Cragg MS
        • Kuroda J
        • Puthalakath H
        • et al.
        Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
        PL oS Med. 2007; 4 (discussion 1690.): 1681-1689
        • Gong Y
        • Somwar R
        • Politi K
        • et al.
        Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas.
        PL oS Med. 2007; 4: e294
        • Pratilas CA
        • Hanrahan AJ
        • Halilovic E
        • et al.
        Genetic predictors of MEK dependence in non-small cell lung cancer.
        Cancer Res. 2008; 68: 9375-9383
        • Yuan TL
        • Cantley LC
        PI3K pathway alterations in cancer: variations on a theme.
        Oncogene. 2008; 27: 5497-5510
        • Erhardt P
        • Cooper GM
        Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL.
        J Biol Chem. 1996; 271: 17601-17604
        • Fan QW
        • Knight ZA
        • Goldenberg DD
        • et al.
        A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.
        Cancer Cell. 2006; 9: 341-349
        • Raynaud FI
        • Eccles S
        • Clarke PA
        • et al.
        Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.
        Cancer Res. 2007; 67: 5840-5850
        • Degtyarev M
        • De Maziere A
        • Orr C
        • et al.
        Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
        J Cell Biol. 2008; 183: 101-116
        • Gozuacik D
        • Kimchi A
        Autophagy and cell death.
        Curr Top Dev Biol. 2007; 78: 217-245
        • Hoyer-Hansen M
        • Jaattela M
        Autophagy: an emerging target for cancer therapy.
        Autophagy. 2008; 4: 574-580
        • Welman A
        • Barraclough J
        • Dive C
        Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2.
        Nat Protoc. 2006; 1: 803-811
        • Girard L
        • Zochbauer-Muller S
        • Virmani AK
        • et al.
        Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering.
        Cancer Res. 2000; 60: 4894-4906
        • Amann J
        • Kalyankrishna S
        • Massion PP
        • et al.
        Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer.
        Cancer Res. 2005; 65: 226-235
        • Shtiegman K
        • Kochupurakkal BS
        • Zwang Y
        • et al.
        Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling.
        Oncogene. 2007; 26: 6968-6978
        • Favata MF
        • Horiuchi KY
        • Manos EJ
        • et al.
        Identification of a novel inhibitor of mitogen-activated protein kinase kinase.
        J Biol Chem. 1998; 273: 18623-18632
        • Klionsky DJ
        • Abeliovich H
        • Agostinis P
        • et al.
        Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
        Autophagy. 2008; 4: 151-175
        • Kabeya Y
        • Mizushima N
        • Ueno T
        • et al.
        LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
        EMBO J. 2000; 19: 5720-5728
        • Mizushima N
        • Yoshimori T
        How to interpret LC3 immunoblotting.
        Autophagy. 2007; 3: 542-545
        • Inukai M
        • Toyooka S
        • Ito S
        • et al.
        Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer.
        Cancer Res. 2006; 66: 7854-7858
        • Tokumo M
        • Toyooka S
        • Ichihara S
        • et al.
        Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer.
        Lung Cancer. 2006; 53: 117-121
        • Maheswaran S
        • Sequist LV
        • Nagrath S
        • et al.
        Detection of mutations in EGFR in circulating lung-cancer cells.
        N Engl J Med. 2008; 359: 366-377
        • Sequist LV
        • Martins RG
        • Spigel D
        • et al.
        First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations.
        J Clin Oncol. 2008; 26: 2442-2449
        • Rhodes N
        • Heerding DA
        • Duckett DR
        • et al.
        Characterization of an Akt kinase inhibitor with potent pharmacodynamic and antitumor activity.
        Cancer Res. 2008; 68: 2366-2374
        • Fan QW
        • Cheng CK
        • Nicolaides TP
        • et al.
        A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma.
        Cancer Res. 2007; 67: 7960-7965