Morphine-induced epidermal growth factor pathway activation in non-small cell lung cancer

Abstract

Background: Epidermal growth factor receptor (EGFR) is coactivated by the μ-opioid receptor (MOR), expressed on non-small cell lung cancer (NSCLC) cells and human lung cancer. We hypothesized that clinically used opioid analgesics that are MOR agonists coactivate EGFR, resulting in growth- and survival-promoting signaling.

Methods: We used H2009, a human adenocarcinoma NSCLC cell line, with constitutive EGFR phosphorylation, which showed increased expression of MOR and the δ-opioid receptor by reverse transcriptase polymerase chain reaction. We used Western immunoblotting, magnetic bead-based Bio-Plex cytokine assay, immunofluorescent staining, BrdU incorporation enzyme-linked immunosorbent assay, and BioCoat™ Matrigel™ invasion assay to examine cell signaling, cytokine expression, colocalization of MOR and EGFR in human lung cancer, and cell proliferation and invasion, respectively.

Results: Similar to epidermal growth factor (EGF), morphine stimulated phosphorylation of EGFR, Akt/protein kinase B (Akt), and mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) signaling in H2009 cells. Opioid receptor (OR) antagonist, naloxone, EGFR tyrosine kinase inhibitor, erlotinib, and silencing of MOR and δ-opioid receptor abrogated morphine- and EGF-induced phosphorylation of signaling, suggestive of OR-mediated coactivation of EGFR. H2009 cells secreted significantly higher levels of cytokines compared with control Beas2B epithelial cells. H2009-conditioned medium stimulated MOR expression in Beas2B cells, suggesting that cytokines secreted by H2009 may be associated with increased OR expression in H2009. We observed colocalization of EGFR and MOR, in human NSCLC tissue. Functionally, morphine- and EGF-induced proliferation and invasion of H2009 cells was ameliorated by naloxone as well as erlotinib.

Conclusion: Morphine-induced phosphorylation of EGFR occurs via ORs, leading to downstream MAPK/ERK, Akt phosphorylation, cell proliferation, and increased invasion. Notably, ORs are also associated with EGF-induced phosphorylation of EGFR. Increased coexpression of MOR and EGFR in human lung cancer suggests that morphine may have a growth-promoting effect in lung cancer.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1. Increased expression of MOR and morphine-induced phosphorylation of EGFR in H2009 NSCLC cells

(A) RT-PCR analysis showing MOR and DOR expression on human lung cancer cell lines H2009 and H460. HaCaT, human keratinocyte cell line known to express high levels of EGFR also shows strong expression of MOR. Loading control for 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, were used as a positive control and HDMEC 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

Figure 2. Naloxone and erlotinib ameliorate morphine- and EGF-induced EGFR, MAPK/ERK, and Akt phosphorylation in H2009 cells

(A) Serum starved H2009 cells were treated with 0.1 microM morphine MS or 20 ng/ml EGF for 10 minutes in the presence or absence of 100 nM naloxone or 1 microM 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. Since erlotinib was dissolved in 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 to PBS 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 anti-human phospho- EGFRTyr1068 at 1:50 dilution, and then incubated with Rhodamine (TRITC) AffiniPure donkey anti-mouse IgG at 1:100. An isotype IgG antibody was used as a control. DAPI was used to co-stain 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 X600. Each figure represents three separate, reproducible experiments. MS: morphine sulfate; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; DAPI: 4’,6-diamidino-2-phenylindole; SF: serum free; GM: growth medium; Nal: naloxone; Erl: erlotinib; DMSO: dimethyl sulfoxide.

Figure 3. Silencing of MOR or DOR inhibits morphine- and EGF-induced signaling

(A) H2009 cells were grown in complete media for 24 hours prior to transfection with human MOR or DOR siRNA (100 nM). RNA was isolated and silencing assessed by RT-PCR. Both MOR and DOR silencing was achieved. Density units for the ratios of MOR or DOR to GAPDH are expressed as mean ± SEM from 3 separate experiments. (B) H2009 cells transfected with MOR or DOR siRNA were serum starved for six hours followed by stimulation with MS or EGF for 10 minutes. MS- and EGF-induced phosphorylation of EGFR, MAPK/ERK and Akt were abrogated in siRNA treated cells compared to 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 to scramble with the same treatment in each graph. These data indicate that MS-induced co-activation of EGFR occurs via opioid receptors, and indicate the necessity of opioid receptors in EGF-induced phosphorylation of EGFR and its downstream pathways. Abbreviations: MOR: mu opioid receptor; DOR: delta opioid receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MS: morphine sulfate; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; MOR: mu opioid receptor.

Figure 4. H2009 conditioned medium increases MOR expression in benign Beas2B bronchoepithelial cells

Beas2B epithelial cells incubated with H2009 conditioned medium for 48h as compared to H2009 blank medium show significantly increased expression of MOR. Density units of MOR:GAPDH are expressed as mean ± SEM of 3 separate experiments. No DOR expression was observed on Beas2B cells incubated with or without H2009 conditioned medium (data not shown). X-axis shows different medium used as follows: Beas2B, Complete Beas2B culture medium BEBM with Singlequotes; H2009 Blank, H2009 culture medium without serum incubated for 48h in a T-75 flask without any cells; H2009 conditioned, H2009 medium incubated with H2009 cells for 48h. Abbreviations: GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MOR: mu opioid receptor

Figure 5. MOR, EGFR, and CD31 co-expression in human NSCLC tumors

Six micron sections of archived human adenocarcinoma (Stage I-III) were co-stained for EGFR (red), MOR (green), and CD31 (blue) as shown in the top row. Images were pseudocolored and overlaid in Adobe Photoshop. Magnification X400. MOR expression is shown by the green arrows. EGFR expression is indicated by the red arrows in the area outlined. EGFR and MOR are co-expressed in tumor tissue but not normal lung tissue as shown in the bottom left panel. CD31 likewise co-localizes with MOR as shown in the bottom middle panel. Most significantly, tumors contained areas where all three markers were co-expressed, as shown in the bottom right panel, indicating endothelial cells are important to the micro-environment of NSCLC tumors and may correlate with MOR and EGFR expression. The 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. Abbreviations: MOR: mu opioid receptor; EGFR: epidermal growth factor receptor; H&E: hematoxylin & eosin.

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 96 a 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 EGF, with or without 0.1 μM naloxone or 1 μM erlotinib for 48 hours. Cell proliferation was measured using BrdU incorporation ELISA. Data are expressed as mean ± SEM from 3 experiments. *P < 0.001 as compared to 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 to 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 EGFR on ORs and vice versa. Abbreviations: MS: morphine sulfate; EGF: epidermal growth factor; NAL: naloxone; ERL: erlotinib; PBS, phosphate buffered saline.