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.
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.
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.
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.
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.
We thank Ms. Carol Taubert for preparation of the document for publication.
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