Lung cancer is the most common cause of cancer deaths worldwide.1,2 Non–small cell lung cancer (NSCLC) comprises approximately 80% of cases; of those, adenocarcinoma is the most common histology.3 The vast majority are diagnosed at an advanced stage, and median survival ranges from 8 to 11 months, indicating a desperate need to further elucidate the molecular pathways driving these tumors and develop new treatments.
Epidermal growth factor receptor (EGFR, also known as erbB-1) is a receptor tyrosine kinase, which has been shown to correlate with poor outcomes in both resected and advanced NSCLC.4–7 The EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib and the anti-EGFR monoclonal antibody cetuximab are used for the treatment of advanced NSCLC,8–11 and mutations imparting significant sensitivity12–14 or resistance15,16 to EGFR TKI therapy are predictive and prognostic biomarkers in NSCLC. Unfortunately, none of these agents is curative, indicating a need to further elucidate mechanisms of resistance to anti-EGFR therapy.
μ-Opioid receptors (MORs) are G-protein coupled receptors (GPCRs) that mediate the analgesic activity of morphine and its congeners to treat pain. In addition to analgesia, morphine/MOR activation stimulates signaling pathways involved in cell proliferation, survival, and migration in a number of cell types.17–24 We showed that morphine stimulates angiogenesis by activating mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) and Akt/protein kinase B (Akt) phosphorylation in human dermal microvascular endothelial cells (HDMECs) and breast cancer progression in mice.22 Morphine activates MAPK/ERK directly and also coactivates vascular endothelial growth factor receptor 2 (VEGFR2) on endothelium.19,20,25 In breast cancer, the growth- and survival-promoting activity of morphine translates into tumor growth, metastasis, and decreased survival in murine models of breast cancer.22,26 Complementary to MOR agonist-induced promotion of tumor growth, the nonselective opioid receptor (OR) antagonist naloxone inhibits human MCF-7 breast cancer cell proliferation and tumor growth in rodents.22,27 The MOR-specific antagonist methylnaltrexone (MNTX) inhibits proliferation and migration of endothelial cells,19 enhances the antitumor effects of the chemotherapeutic agent 5-fluorouracil in breast, lung, and colon cancer cell lines, and synergizes with bevacizumab and 5-fluorouracil to inhibit VEGF-induced angiogenesis.20,28
A recent demonstration of inhibition of Lewis lung carcinoma (LLC) in MOR knockout mice as compared with wild-type mice further exemplified the significance of MOR in lung cancer.23 Expression of the immunoreactive opioid peptides β-endorphin, enkephalin, and dynorphin, and the presence of high-affinity membrane receptors for μ-, δ-, and κ-opioid receptors (MOR, DOR, and KOR) on diverse small cell lung carcinoma (SCLC) and NSCLC cell lines were demonstrated on the basis of ligand binding studies29,30 2 decades ago. Subsequent studies showed that methadone inhibited lung cancer cell growth by promoting apoptosis via stimulation of MAPK-phosphatase, inactivation of MAPK, and suppression of bcl-2 in low-concentration bombesin-secreting SCLC and NSCLC cells but not in cells secreting higher concentrations of bombesin.31 Importantly, in the same study, morphine and the MOR-specific agonist [D-Ala2, N-MePhe4, Glu-ol]-enkephalin (DAMGO) stimulated MAPK/ERK phosphorylation whereas methadone inhibited MAPK/ERK phosphorylation. The authors suggested that methadone acted via a non–OR-mediated mechanism, but did not provide an explanation for morphine- and DAMGO-induced MAPK/ERK phosphorylation. The presence of MOR and DOR has been shown in human lung cancers in vivo using positron emission tomography scanning.32 These authors showed the presence of binding sites for the DOR-selective antagonist 11C-methylnaltrindole (11C-MeNTI) and the MOR-specific agonist 11C-carfentanil (11C-CFN) in patients with small cell, squamous cell, and adenocarcinoma. Increased binding of 11C-MeNTI and 11C-CFN was observed in all lung tumors compared with noncancerous lung. These studies clearly demonstrate the presence of ORs and highlight their significance in lung cancer growth. However, the mechanism(s) of OR-mediated lung cancer progression is unclear.
MOR agonists also transactivate receptor tyrosine kinases, including EGFR,33–35 but this has not been examined in NSCLC. Because MOR is activated by clinically relevant concentrations of opioids and EGFR signaling is critical in the malignant phenotype of lung adenocarcinomas, we hypothesize that ORs in NSCLC coactivate EGFR and downstream signaling pathways that promote proliferation and survival. We used NCI-H2009 human adenocarcinoma lung cancer cells because of their resistance to therapy and increased expression of MOR compared with other NSCLC cell lines. We show that morphine stimulates the phosphorylation of EGFR, MAPK/ERK, and Akt via MOR, and the effect is attenuated by the OR antagonist naloxone as well as by the EGFR inhibitor erlotinib.
METHODS
Cell Culture
The human NCI lung carcinoma cell lines H2009 and H460 were obtained from ATCC (Manassas, VA) and maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum. Beas2B, an adenovirus-12 SV40 immortalized human bronchoepithelial cell line, was obtained from ATCC and maintained in BEBM (Invitrogen) supplemented with SingleQuots (Cambrex Bio Science, Walkersville, MD). HDMECs were cultured from neonatal human foreskin, maintained in media containing 20% male human serum as described previously,36 and used between passages 4 to 8. The human epidermal keratinocyte cell line, HaCaT, was obtained from Carol A. Lange, PhD, at the University of Minnesota. All cells were incubated at 37°C and 5% CO2.
Reverse Transcriptase Polymerase Chain Reaction
RNA was isolated from H460, H2009, and HDMECs using TRIzol (Invitrogen), and reverse transcriptase polymerase chain reaction (RT-PCR) was performed using Taq DNA polymerase (Continental Laboratory Products, San Diego, CA). The following primers were used:
- MOR (440 bp): forward: GGT ACT GGG AAA ACC TGC TGA AGA TCT GTG.
- reverse: GGT CTC TAG TGT TCT GAC GAT TCG AGT GG.
- DOR (365 bp): forward: ATC TTC ACC CTC ACC ATG ATG.
- reverse: CGG TCC TTC TCC TTG GAA CC.
- GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (452 bp): forward: GAA GGT GAA GGT CGG AGT C.
- reverse: GAA GAT GGT GAT GGG ATT TC.
- DNA samples were visualized by 2% agarose gel electrophoresis.
Western Immunoblotting
H2009 cells were grown in regular growth media for 24 hours, incubated in 0.5% reduced serum media overnight, and whole cell lysates were prepared after the treatment incubations (Figs. 1–6). Lysates (100 μg protein) resolved on a 3% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel were transferred to a polyvinylidene fluoride membrane (Immobilon; Millipore, Bedford, MA) and probed for mouse antihuman phospho-EGFRTyr1173 (Millipore) at 1:2000 dilution or total EGFR, rabbit antihuman phospho-p42/44 MAPK (ERK1/2)Thr202/Tyr204, or total MAPK/ERK (Cell Signaling, Danvers, MA) at 1:500, and rabbit antihuman phospho-AktThr308 or total Akt (Cell Signaling) at 1:500. After washing, membranes were incubated in the appropriate species-specific secondary antibody linked to alkaline phosphatase for 1 hour at room temperature. Chemiluminescent signals were detected using the ECF System (Amersham Biosciences, Piscataway, NJ), and images were acquired using a Storm 860 Phosphoimager (Molecular Dynamics, Sunnyvale, CA).
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Figure 1: Increased expression of μ-opioid receptor (MOR) and morphine-induced phosphorylation of epidermal growth factor receptor (EGFR) in H2009 non–small cell lung cancer cells. A, Reverse transcriptase polymerase chain reaction analysis showing MOR and δ-opioid receptor (DOR) expression on human lung cancer cell lines H2009 and H460. HaCaT, a human keratinocyte cell line known to express high levels of EGFR also shows strong expression of MOR. Loading control for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown in the bottom row. Density units relative to GAPDH are shown. B, Western immunoblotting showing constitutive phosphorylation of EGFR on H2009 cells. HaCaT cells were used as a positive control and human dermal microvascular endothelial cells (HDMECs) as a negative control. Density units for phospho-EGFR relative to total EGFR are shown. C, Morphine- and EGF-induced time-dependent phosphorylation of EGFR on H2009 grown overnight in serum-reduced conditions. Density units for phosph-EGFR relative to total EGFR for each treatment are shown. *P < 0.001 and **P < 0.01, as compared with unstimulated (US) 0.5% serum. Each figure represents 3 separate, reproducible experiments.
Figure 2: Naloxone and erlotinib ameliorate morphine- and epidermal growth factor (EGF)-induced epidermal growth factor receptor (EGFR), mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), and Akt/protein kinase B (Akt) phosphorylation in H2009 cells. A, Serum-starved H2009 cells were treated with 0.1 μM morphine morphine sulfate (MS) or 20 ng/mL EGF for 10 minutes in the presence or absence of 100 nM naloxone or 1 μM erlotinib. MS- and EGF-induced EGFR, MAPK/ERK, and Akt phosphorylation were abrogated by both naloxone and erlotinib. Respective total protein levels did not change. Because erlotinib was dissolved in dimethyl sulfoxide (DMSO), H2009 cells were stimulated with MS and EGF containing the same concentration of DMSO as erlotinib. Ratios of density units for each phospho- to total protein are shown. $P < 0.05, *P < 0.01, and **P < 0.001, as compared with phosphate-buffered saline in each graph. B, H2009 cells were grown on glass slides for 24 hours in regular growth media, serum starved overnight, and treated with morphine or EGF in the presence or absence of naloxone for 10 minutes (similar to A). Cells were incubated with mouse antihuman phospho-EGFRTyr1068 at 1:50 dilution, and then incubated with Rhodamine (TRITC) AffiniPure donkey antimouse immunoglobulin (Ig)G at 1:100. An isotype IgG antibody was used as a control. DAPI (4′,6-diamidino-2-phenylindole) was used to costain the nuclei. Please note the inset in EGF 10′ showing intense granular staining of phospho-EGFR over the entire cell. In contrast, EGF + Nal 10′ shows red staining of phospho-EGFR confined to one end and overall weak staining for phospho-EGFR. Magnification, ×600. Each figure represents 3 separate, reproducible experiments. SF = serum free; GM = growth medium; Nal = naloxone; Erl = erlotinib.
Figure 3: Silencing of μ-opioid receptor (MOR) or δ-opioid receptor (DOR) inhibits morphine- and epidermal growth factor (EGF)-induced signaling. A, H2009 cells were grown in complete media for 24 hours before transfection with human MOR or DOR siRNA (100 nM). RNA was isolated and silencing assessed by reverse transcriptase polymerase chain reaction. Both MOR and DOR silencing was achieved. Density units for the ratios of MOR or DOR to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are expressed as mean ± SEM from 3 separate experiments. B, H2009 cells transfected with MOR or DOR siRNA were serum starved for 6 hours followed by stimulation with morphine sulfate (MS) or EGF for 10 minutes. MS- and EGF-induced phosphorylation of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), and Akt/protein kinase B (Akt) was abrogated in siRNA-treated cells compared with untransfected cells. Scramble siRNA was used as a negative control and had no effect. Density units of phospho- to total protein for each signaling pathway are shown as mean ± SEM of 3 different experiments. $P < 0.05, *P < 0.01, and **P < 0.001, as compared with scramble with the same treatment in each graph. These data indicate that MS-induced coactivation of EGFR occurs via opioid receptors, and indicate the necessity of opioid receptors in EGF-induced phosphorylation of EGFR and its downstream pathways. PBS = phosphate-buffered saline.
Figure 4: H2009-conditioned medium increases μ-opioid receptor (MOR) expression in benign Beas2B bronchoepithelial cells. Beas2B epithelial cells incubated with H2009-conditioned medium for 48 hours as compared with H2009 blank medium show significantly increased expression of MOR. Density units of MOR:GAPDH (glyceraldehyde 3-phosphate dehydrogenase) are expressed as mean ± SEM of 3 separate experiments. No δ-opioid receptor (DOR) expression was observed on Beas2B cells incubated with or without H2009-conditioned medium (data not shown). The x-axis shows different mediums used as follows: Beas2B = complete Beas2B culture medium BEBM with SingleQuot; H2009 blank = H2009 culture medium without serum incubated for 48 hours in a T-75 flask without any cells; H2009 conditioned = H2009 medium incubated with H2009 cells for 48 hours.
Figure 5: μ-Opioid receptor (MOR), epidermal growth factor receptor (EGFR), and CD31 coexpression in human non–small cell lung cancer (NSCLC) tumors. Six-micrometer sections of archived human adenocarcinoma (stages I–III) were costained for EGFR (red), MOR (green), and CD31 (blue) as shown in the top row. Images were pseudo-colored and overlaid in Adobe Photoshop. Magnification ×400. MOR expression is shown by the green arrows. EGFR expression is indicated by the red arrows in the area outlined. EGFR and MOR are coexpressed in tumor tissue but not normal lung tissue as shown in the bottom left panel. CD31 likewise colocalizes with MOR as shown in the bottom middle panel. Most significantly, tumors contained areas where all 3 markers were coexpressed, as shown in the bottom right panel, indicating endothelial cells are important to the microenvironment of NSCLC tumors and may correlate with MOR and EGFR expression. The hematoxylin & eosin (H&E) image is a histologically representative area of the immunofluorescent images. Images shown represent 8 tumors stained. The images above were obtained from a stage IB adenocarcinoma.
Figure 6: Morphine stimulates the proliferation and invasiveness of H2009 cells. A, H2009 cells were plated at a density of 2 × 103 per well in a 96-well plate and incubated in regular growth media overnight followed by serum starvation for another night. Cells were then incubated with 0.1 μM morphine or 20 ng/mL epidermal growth factor (EGF), with or without 0.1 μM naloxone or 1 μM erlotinib for 48 hours. Cell proliferation was measured using BrdU incorporation enzyme-linked immunosorbent assay. Data are expressed as mean ± SEM from 3 experiments. *P < 0.001 as compared with phosphate-buffered saline (PBS). B, H2009 cells (1 × 105 per chamber) were allowed to migrate in Matrigel-coated invasion chambers for 24 hours in the treatment conditions described for A. Cells migrated per field of view (average of 4 fields per well) are expressed as a mean ± SEM from 3 separate experiments. $P < 0.05, *P < 0.01, and **P < 0.001, as compared with PBS. Morphine-induced proliferation and invasion were blocked by naloxone as well as erlotinib. Likewise, EGF-induced proliferation and invasion were blocked by either naloxone or erlotinib, indicative of the dependence of epidermal growth factor receptor on opioid receptors and vice versa. MS = morphine sulfate; NAL = naloxone; ERL = erlotinib.
Densitometry
Quantification of immunoblotting and RT-PCR bands were obtained by densitometric analysis using ImageJ Software (National Institutes of Health, Bethesda, MD).
siRNA Transfection
H2009 cells were grown in regular growth media overnight, then transfected with 100 nM human MOR siRNA, DOR siRNA, or scramble siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA) in Optimem (Invitrogen) using siPORT lipid transfection agent (Ambion, Austin, TX). Regular growth media was added after 8 hours of transfection and cells were incubated overnight. Both siRNAs comprised a pool of 3 target-specific siRNAs. RNA was isolated as described above and RT-PCR was performed to assess knockdown. DNA samples were visualized by 2% agarose gel electrophoresis to ensure gene knockdown using the primers described above. For immunoblotting experiments, transfected cells were serum starved for 6 hours and then stimulated with morphine or epidermal growth factor (EGF) as described below.
Cytokine Analysis
Subconfluent cultures of H2009 and Beas2B cells were incubated for 48 hours in their respective media, and the conditioned media was snap frozen and stored at −80°C until assayed. Control media was prepared in parallel by incubating media without cells in identical flasks. Magnetic bead–based Bio-Plex assays (BioRad Laboratories, Inc., Hercules, CA) were used to determine the concentration of the angiogenic factors as described previously.37 Briefly, color-coded magnetic beads are coupled to an antibody unique to the marker analyzed and dyed with 2 fluorophores (classification dyes), which absorb maximally at 635 nm and emit at 2 distinct wavelengths. The reporter dye is a third fluorophore, phycoerythrin, which emits at a third distinct wavelength and has a maximal absorption at 532 nM. The detector unit consists of a flow cell that enables the magnetic beads to travel in a single file (laminar flow) through a region illuminated by a pair of lasers. Values shown are the mean ± SEM of 6 separate experiments. P values were determined by analysis of variance.
Beas2B Cell Treatment With H2009-Conditioned Medium
H2009 cells were incubated with serum-free H2009 medium for 48 hours to prepare H2009-conditioned medium. Beas2B cells plated overnight in complete Beas2B culture medium were growth factor–starved and incubated for an additional night. The next day, Beas2B cells were incubated for 48 hours in growth factor–free medium or in 50% growth factor–free medium + 50% H2009 serum-free medium or in 50% growth factor–free medium + H2009-conditioned medium. RNA was isolated for MOR and DOR expression, and RT-PCR was performed as described above. Cell lysates were examined for autophosphorylation of Beas2B cells by Western immunoblotting as described above.
Immunofluorescent Microscopy of H2009 Cells
H2009 cells were grown on glass slides for 24 hours in regular growth media and serum starved overnight. Cells were treated with 0.1 μM morphine or 100 ng/mL EGF for 10 minutes with or without preincubation with 10 nM naloxone for 10 minutes. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 on ice. Cells were then blocked with 3% bovine serum albumin and incubated with mouse antihuman phospho-EGFRTyr1068 (Cell Signaling) primary antibody at 1:50 dilution, followed by incubation with Rhodamine (TRITC) AffiniPure donkey antimouse immunoglobulin (Ig)G (Jackson ImmunoResearch, West Grove, PA) secondary antibody at 1:100, and 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) at 1:50,000 to costain the nucleus during the last 10 minutes of incubation. An isotype IgG antibody (Jackson ImmunoResearch) was used in parallel as a control. Fluorescent images were obtained using an Olympus 1X70 microscope fitted with an Olympus DP70 digital camera at ×600 (Olympus America, Melville, NY).
Immunofluorescent Staining of Human Adenocarcinoma Tissue
Archived, paraffin-embedded human lung adenocarcinomas were obtained from the VA Medical Center, Minneapolis, MN. Six-micrometer sections were costained with the following primary antibodies: goat antihuman CD31 (Santa Cruz Biotechnology) at 1:50 dilution to label blood vessels, mouse antihuman total EGFR (Abcam, Cambridge, MA) at 1:100, and rabbit antihuman MOR (Chemicon International, Temecula, CA) at 1:100, followed by staining with species-specific secondary antibodies labeled with Cy2 (CD31), Cy3 (EGFR), and Cy5 (MOR) (Jackson ImmunoResearch). Images were obtained using an Olympus BX50 upright epifluorescent microscope at ×200 (Olympus America). MOR was pseudo-colored green, EGFR red, and CD31 blue using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). Images were overlaid using Adobe Photoshop.
Proliferation Assay
Cell proliferation was measured using BrdU cell proliferation enzyme-linked immunosorbent assay (Roche, Indianapolis, IN) according to the manufacturer's instructions, as described previously.24 Briefly, 2000 cells per well were plated on a 96-well plate in complete culture medium for H2009 cells and incubated overnight. The next day, cells were incubated with serum-free medium overnight, followed by incubation with different agonists and antagonists for 48 hours (Fig. 6) (morphine sulfate 0.1 μM; naloxone, 0.1 μM; erlotinib, 1 microM; and EGF, 20 ng/mL). BrdU enzyme-linked immunosorbent assay was performed using peroxidase-conjugated anti-BrdU antibodies. Color developed was measured at 370 nm with 492 nm as the reference wavelengths.
Invasion Assay
H2009 cell invasion was assessed using 24-well BioCoat™ Matrigel™ invasion chambers (Becton Dickinson Biosciences, Bedford, MA) with 8-μm pore size according to the manufacturer's instructions. Serum-free medium supplemented with morphine or EGF, with and without naloxone or erlotinib, was added to the bottom chamber of the well in a 24-well cluster. Serum-free medium containing 1 × 105 H2009 cells was added to the top chamber of the inserts. Cells were allowed to migrate for 24 hours. Cells remaining on the upper side of the membrane were removed with a cotton-tipped applicator. Cells that had migrated through the membrane were fixed in methanol and stained with crystal violet. Cells were counted in a blinded manner in 4 random areas per membrane at a magnification of ×100 using an Olympus CK2 inverted microscope (Olympus America).
Statistical Analysis
All statistical analyses were performed using Prism software (GraphPad Prism Inc., San Diego, CA). Unpaired Student t tests or analysis of variance followed by Tukey post hoc test were performed to determine the significance between different conditions. A P < 0.05 was considered to be statistically significant.
RESULTS
MOR and EGFR Are Upregulated in NSCLC Cell Lines, and Morphine Coactivates EGFR
H2009, an adenocarcinoma cell line resistant to therapy, showed significantly higher expression of MOR and DOR as compared with H460, a large cell lung carcinoma cell line (Fig. 1A). Therefore, H2009 was selected for further experiments. Increased MOR expression was accompanied by significantly higher constitutive phosphorylation of EGFR similar to that of HaCaT cells, a human keratinocyte cell line, as compared with HDMECs (Fig. 1B). Both morphine and EGF stimulated the phosphorylation of EGFR in a time-dependent manner (Fig. 1C). These data show correlative expression of increased MOR and constitutively phosphorylated EGFR, and morphine-induced phosphorylation of EGFR, suggestive of MOR–EGFR cross-talk.
Morphine and EGF Induced EGFR Coactivation, and Downstream Signaling Are Abrogated by Naloxone and Erlotinib
Western immunoblotting and densitometric analysis of protein bands showed that morphine-induced EGFR phosphorylation in H2009 cells was accompanied by a significant increase in phosphorylation of MAPK/ERK and Akt (Fig. 2A). Naloxone, a nonselective OR antagonist, significantly inhibited morphine- as well as EGF-induced phosphorylation of EGFR, MAPK/ERK, and Akt (Fig. 2A). Activation of these pathways by morphine as well as EGF was significantly ameliorated by erlotinib, an inhibitor of EGFR. Immunofluorescent analysis of the phosphorylation of EGFR by EGF and morphine also showed increased staining of membrane-bound phospho-EGFR after 10 minutes of incubation (Fig. 2B). Dense red punctate staining of phospho-EGFR was seen over the entire cell body after 10 minutes of treatment with EGF (inset in the box), which was reduced in cells pretreated with naloxone. Morphine-induced staining was more densely associated with the cell membrane and sparsely with the nuclei and cytoplasm, and was inhibited by naloxone. These data suggest OR and EGFR cross-talk that influences downstream signaling pathways, MAPK/ERK, and Akt upon stimulation with morphine or EGF.
Morphine-Induced Coactivation of EGFR Occurs via MOR and DOR
Silencing of MOR and DOR was achieved by transfecting H2009 cells with a cocktail of 3 different siRNA sequences for MOR or DOR (Fig. 3A). siMOR and siDOR silenced the expression of MOR and DOR, respectively, on H2009 cells but scramble sequence did not alter the MOR and DOR expression. Western immunoblotting and densitometric analysis of protein bands showed that silencing of MOR and DOR significantly abrogated morphine- and EGF-induced phosphorylation of EGFR, MAPK/ERK, and Akt as compared with H2009 cells treated with scramble siRNA (Fig. 3B). These data suggest that activation of EGFR and downstream MAPK/ERK and Akt pathways is dependent on activation of MOR and DOR.
NSCLC Cells Secrete Prosurvival and Proangiogenic Factors
We next examined cytokines secreted by H2009 cells compared with benign Beas2B epithelial cells (Table 1) to determine whether the increased MOR and DOR expression and EGFR autophosphorylation is attributable to the secretion of cytokines by H2009 cells. Hepatocyte growth factor (HGF), the only known ligand for mesenchymal-epithelial transition factor (c-MET), a tyrosine kinase overexpressed in the majority of NSCLC involved in cell proliferation, survival, migration, and carcinogenesis38–40 was significantly higher in H2009-conditioned media (P = 0.0023). c-MET crosstalk with EGFR has been demonstrated,39 and indeed, EGFR inhibition has been shown to lead to abrogation of HGF-induced c-MET activation, thus mitigating HGF-induced migration.41 Additionally, cMET amplification has been shown to impart resistance to the EGFR TKI gefitinib in vitro.42 Interleukin-8 and VEGF were significantly higher in H2009 cells as compared with Beas2B-conditioned medium (P < 0.001). VEGF upregulates MOR expression in endothelial cells25; this is a plausible explanation of the MOR upregulation observed in H2009 cells (Fig. 1) and in NSCLC tissue (Fig. 5). Antiangiogenic therapy in lung cancer has not been therapeutic, despite its success in animal models,43–45 perhaps because of a self-supportive and proangiogenic environment created by the lung cancer cells. Granulocyte colony-stimulating factor is also overexpressed in H2009 medium as compared with Beas2B medium (P = 0.0004). Granulocyte colony-stimulating factor seems to have a role in angiogenesis, and imparts resistance to bevacizumab when given exogenously in a mouse model.46 Platelet endothelial cell adhesion molecule-1, believed to be involved in angiogenesis and endothelial cell migration46 and follistatin, which is overexpressed in patients with NSCLC,47 were also markedly higher in H2009 cell supernatants (P = 0.0001). Together, these data support the hypothesis that these adenocarcinoma cells possess the ability to self-create a proangiogenic, growth-promoting, and prosurvival environment, by inducing the expression of ORs and constitutive phosphorylation of EGFR.
Table 1: H2009 Cells Secrete Growth-Promoting Cytokines
Furthermore, incubating Beas2B cells in H2009-conditioned medium for 48 hours resulted in significantly increased MOR expression (Fig. 4), but did not stimulate DOR expression or autophosphorylation of EGFR (data not shown). Of note, Beas2B cells did not show any constitutive expression of DOR or autophosphorylation of EGFR. Therefore, the tumor microenvironment seems to be more conducive to MOR expression in nonmalignant cells.
MOR, EGFR, and CD31 Are Coexpressed by Human NSCLC Tumors
Correlative to our in vitro observations of increased MOR and EGFR expression on H2009 cells and increased levels of proangiogenic and survival-promoting cytokines, we observed increased coexpression of EGFR and MOR in the tumor region of resected human lung adenocarcinomas compared with normal lung tissue (Fig. 5). EGFR and MOR expression colocalized with CD31-positive as well as CD31-negative cells, suggesting that MOR and EGFR expression increases on vascular endothelial cells and on tumor cells. Green staining for vascular endothelium (top left-hand panel) shows MOR coexpression (bottom left-hand panel) in turquoise color. Merged images of CD31, MOR, and EGFR show white staining suggestive of EGFR and MOR coexpression on CD31-positive endothelial cells (bottom right panel); magenta staining shows the coexpression of MOR with EGFR on nonvascular cells. A tumor microenvironment replete with increased cytokines and growth factors may be involved in the increased vascularity that correlates with increased expression of MOR and EGFR in human lung tumors.
Morphine- and EGF-Induced Proliferation and Invasion of H2009 Is Mediated by OR(s) and EGFR
EGF and morphine significantly stimulated the proliferation and invasiveness of H2009 cells as compared with phosphate-buffered saline–treated cells (Fig. 6, A and B; P < 0.001 and 0.05, respectively). These functional effects of morphine and EGF were significantly inhibited by pretreatment of cells with OR antagonist, naloxone or EGFR inhibitor, erlotinib. These data demonstrate the functional dependence of EGFR on ORs and vice versa. Correlatively, these observations provide the functional significance of morphine-induced activation of growth-promoting signaling pathways described above.
DISCUSSION
The influence of opioids on tumor biology has remained elusive, and limited data argue for both tumor-inhibitory and tumor-promoting effects of opioids.22,23,30,31 However, emerging literature strongly suggests that MOR agonist opioids at clinically used doses induce endothelial and tumor cell proliferation, enhance survival, and migration both in vitro and in vivo.18,19,27 Our data demonstrate that (1) activation of ORs by analgesic morphine coactivates EGFR in highly resistant NSCLC cells, (2) ORs are involved with EGF-induced EGFR, MAPK/ERK and Akt phosphorylation, proliferation and invasiveness, and (3) the self-supportive tumor microenvironment created by NSCLC cells may be associated with increased expression of MOR on tumor cells. Therefore, our data provide an insight into the complex mechanism(s) that may impart resistance to therapy in difficult-to-treat lung cancer.
EGFR is a critical target in the treatment of advanced NSCLC; however, EGFR-targeted therapies are not curative, indicating a need to identify additional targets to block cancer progression. In this regard, we identified ORs as potential contributing factors to EGFR TKI resistance. We show that ORs are expressed on human NSCLC cells both in vitro and in vivo. This is in agreement with previous work demonstrating increased MOR expression in human lung cancers as detected by positron emission tomography scanning,32 opioid agonist and antagonist binding,30 and by immunohistochemistry.23 Our observations are in agreement with a recent study showing overexpression of MOR on several human NSCLC adenocarcinoma and bronchioalveolar carcinoma cell lines and human lung adenocarcinoma and bronchioalveolar carcinoma tissue.23 However, herein, the finding of increased coexpression of EGFR with overexpression of MOR in human lung adenocarcinoma tissue specimens is a novel finding. Correlative to EGFR and MOR overexpression in the cancerous lung adenocarcinoma, we observed autophosphorylation of EGFR without external stimulation in H2009 cells. We believe that the tumor microenvironment replete with proinflammatory and growth- and survival-promoting cytokines stimulates the expression of MOR in lung cancer cells as well as in the cancerous tissue, observed by us. This is further supported by the markedly increased expression of cytokines in the culture medium of H2009 cells herein. In earlier studies, we observed that VEGF increased MOR gene and protein expression several-fold in mouse retinal microvascular endothelial cells.25 It is therefore conceivable that the highly enriched cytokine microenvironment in the tumor and that observed by us in H2009 cell culture medium orchestrates MOR expression in the tumor and H2009 cells, respectively.
Indeed, incubation of benign Beas2B lung epithelial cells with H2009-conditioned medium, which led to increased MOR expression on Beas2B cells, supports our hypothesis that increased secretion of cytokines by NSCLC enhance MOR expression on H2009 and in lung tumors. Whether this increased MOR expression is transformative remains to be investigated. Beas2B cells did not show constitutive DOR expression or autophosphorylation of EGFR, nor were they stimulated by H2009-conditioned medium. It is likely that long-term stimulation with cytokines such as those secreted by H2009 may be required to cause EGFR autophosphorylation in benign cells or transformation to tumor cell phenotype may be a prerequisite to cytokine-induced DOR and/or EGFR autophosphorylation. Thus, NSCLC cells are able to maintain a self-promoting and self-sustaining environment, perhaps via overexpression of MOR and constitutive activation of EGFR observed in H2009 cells and in human lung adenocarcinoma.
The constitutive phosphorylation of EGFR is further increased upon stimulation with morphine in H2009 cells, which is accompanied by phosphorylation of downstream MAPK/ERK and Akt signaling pathways analogous to EGF-induced signaling. Our observations are in agreement with morphine-induced and highly selective MOR agonist DAMGO-induced MAPK/ERK phosphorylation in NSCLC cells shown more than a decade ago.31 Activation of these pathways by morphine translates into increased proliferation and invasiveness of H2009 cells, suggestive of a growth-promoting effect of morphine in lung cancer. These signaling and functional activities of morphine in H2009 cells are reminiscent of morphine-induced phosphorylation of MAPK/ERK, Akt, and VEGFR2, and proliferation, migration, and survival of endothelial cells observed by us, and others.19–22,25 Our observations on morphine-induced proliferation of H2009 NSCLC cells are further supported by a recent study showing significantly increased proliferation of LLC cells in response to morphine and a highly selective MOR agonist peptide, DAMGO.23 Together, these observations demonstrate that morphine stimulates lung cancer cell proliferation and invasion by activating EGFR, MAPK/ERK, and Akt signaling pathways.
Inhibition of morphine-induced EGFR, Akt, and MAPK/ERK phosphorylation by naloxone implicates ORs in this phenomenon. Silencing of MOR as well as DOR on H2009 cells results in abrogation of morphine and EGF-induced signaling, suggesting an integral role of MOR and DOR in lung cancer progression. These observations on H2009 cells complement the findings on inhibition of invasion and colony formation in MOR-silenced LLC cells and a significant inhibition in tumor burden and metastases in MOR knockout mice as compared with wild-type mice with subcutaneously growing LLC.23 Invasion of LLC was also inhibited by a highly specific MOR antagonist, MNTX, in this study. Moreover, inhibition of morphine-induced EGFR, Akt, and MAPK/ERK phosphorylation by erlotinib and in the MOR/DOR-silenced H2009 noted by us, supports a mechanism whereby morphine-induced stimulation of growth- and survival-promoting signaling requires cross-talk between MOR/DOR and EGFR. The observations on MOR overexpression and inhibition of its activity pharmacologically by MNTX or erlotinib may be critical in resistant, difficult-to-treat lung cancer. Taken together, these data suggest that the addition of morphine to an NSCLC microenvironment with upregulated MOR may increase growth-promoting and prosurvival signaling directly via ORs and by coactivating EGFR.
The observed inhibition of morphine- and EGF-induced signaling by silencing DOR may be attributable to the formation of MOR and DOR heterodimers,48,49but this requires further investigation. Due to severe pain accompanying advanced stage lung cancer, MOR seems to be highly critical, because of the use of opioid analgesics that act via MOR. However, our observations on the abrogation of morphine- and EGF-induced signaling by silencing of DOR may be important in developing DOR antagonists to inhibit the unwanted tumor growth-promoting activity of opioids without compromising analgesia. Naltrindole, a classic DOR antagonist, was shown to inhibit the Akt survival-promoting pathway and modulation of several Akt-dependent genes and the downstream effectors, glycogen synthase kinase-3β and Forkhead transcription factors AFX and FKHR, in SCLC cells.50 In this study, naltrindole also induced apoptosis in SCLC cells by inhibiting Akt signaling. Thus, DOR may provide additional targets to abrogate the unwanted effect of morphine on lung cancer progression.
Although the exact mechanism by which OR inhibition abrogates morphine- and EGF-induced EGFR, Akt, and MAPK/ERK phosphorylation has not yet been elucidated in NSCLC, our model suggests that EGFR coactivation by MOR occurs intracellularly as it does in a number of other cell types. EGFR transactivation occurs via intracellular, zinc-dependent ADAM (a disintegrin and metalloprotease) family protease cleavage of membrane-bound EGF-like ligands such as heparin binding-EGF, amphiregulin, and transforming growth factor-α, in a variety of cell types.35,49,51 Moreover, EGFR and MAPK can be transactivated by MOR stimulation via a calmodulin-dependent mechanism in human embryonal kidney cells.33 Conversely, EGF has been shown to phosphorylate MOR via GRK2 (G protein receptor kinase 2) in human embryonal kidney cells.52 Cross-talk between EGFR and GPCRs has been suggested to be src and ERK dependent.53 In this study, inhibition of GPCR or EGFR inhibited the activity of the other. This is a plausible explanation for our observation that naloxone and OR silencing or erlotinib attenuated EGF- or morphine-induced EGFR phosphorylation. Therefore, it is likely that MOR-specific antagonists will have an inhibitory effect on lung cancer as suggested by Mathew et al.23 These data provide insight into a potential means to overcome resistance to anti-EGFR therapy, which is made particularly attractive with the recent development of peripherally only acting MOR antagonists such as MNTX that do not antagonize analgesia by morphine.
Recently, Singleton et al.21 reported synergy between MNTX and the mTOR inhibitors rapamycin and temsirolimus in inhibiting VEGF-induced human pulmonary vein endothelial cell proliferation, migration, and angiogenesis. Similar to therapies for other cancers, multitargeted therapies addressing both the endothelium and tumor cells seem to be more effective than monotherapy.54 These recent observations are further supported by earlier observations that MNTX can have an inhibitory effect on VEGFR2 and EGFR activity on endothelial and lung cancer cells, respectively,19,20,23 and by our data showing colocalization of EGFR and MOR on CD31-positive endothelial cells in addition to tumor cells.
Based on our observations, we speculate important clinical implications for the involvement of ORs in NSCLC: (1) upregulation of MOR may have adverse effects on the promotion of tumor growth, (2) opioid analgesics that are MOR agonists such as morphine may inadvertently promote cancer progression when used for analgesia, and (3) peripherally acting OR antagonism can be targeted to develop adjunctive therapy to treat lung cancer.
DISCLOSURES
Name: Naomi Fujioka, MD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Naomi Fujioka has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Julia Nguyen.
Contribution: This author helped conduct the study.
Attestation: Julia Nguyen has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Chunsheng Chen.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Chunsheng Chen has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Yunfang Li.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Yunfang Li has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Teena Pasrija.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Teena Pasrija has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Gloria Niehans.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Gloria Niehans has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Katherine N. Johnson.
Contribution: This author helped conduct the study.
Attestation: Kathryn N. Johnson has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Vinita Gupta, PhD.
Contribution: This author helped conduct the study.
Attestation: Vinita Gupta has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Robert A. Kratzke, MD.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Attestation: Robert A. Kratzke has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Kalpna Gupta, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Kalpna Gupta has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
ACKNOWLEDGMENTS
We thank Ms. Carol Taubert for preparation of the document for publication.
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