Research ArticleGastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib
Introduction
The bombesin-like peptides, including gastrin-releasing peptide (GRP), have been shown to exert multiple functions on cell growth, proliferation, and survival as well as to have involvement in physiological and pathological processes. GRP and other members of the bombesin-like peptide family are known to promote proliferation and growth of Swiss 3T3 fibroblasts [1], to stimulate release of gastrin from G cells in gastrointestinal tract, to promote fetal lung development and lung injury repair [2], and to stimulate proliferation and growth of bronchial epithelial cells and cancer cells [3]. GRP receptor (GRPR) is more frequently expressed in the bronchial epithelium of women than that of men in the absence of tobacco smoking, and the expression of GRPR is activated earlier in women in response to tobacco exposure [4]. Given that tobacco smoking is the most important risk factor for development of lung cancer, effects of GRP on bronchial epithelial cells could contribute significantly to lung tumorigenesis. In addition, GRP is secreted by both small cell lung carcinoma (SCLC) cells and NSCLC cells that express receptors for this peptide [5], [6], [7]. Increasing lines of evidence show that GRP and other bombesin-like peptides can promote cell growth in both NSCLC cells and SCLC cells [6], [8]. The production of GRP by NSCLC cells and expression of its receptor in these cells strongly suggest that an autocrine or paracrine loop plays a role in cell growth and proliferation. However, the role of GRP in mediating the response of NSCLC cells to chemotherapy and biological therapy has not been elucidated.
The receptor for GRP is a member of the G-protein-coupled receptor family [9]. Whereas signal transduction pathways have been widely explored in relation to GRP-induced cellular proliferation and growth, few studies have investigated GRP-induced intracellular events related to the resistance of NSCLC cells to therapy. Previous studies suggest that GRP induces cell proliferation and growth through different signaling pathways in different cell lines. While GRP treatment results in the activation of phospholipase C and Ca++ influx in 3T3 fibroblasts [10] and increased intracellular Ca2+ and cAMP in pancreatic adenocarcinoma cells [11], it causes activation of protein kinase C and p38 kinase in duodenal cancer cells [12]. On the other hand, GRP stimulates the activation of mitogen-activated protein kinase (MAPK) in NSCLC, head and neck carcinoma cells, and rat fibroblasts [13], [14], [15]. GRP stimulates phosphorylation of tyrosine kinase receptors such as epidermal growth factor receptor (EGFR) prior to the MAPK activation in head and neck carcinoma cells [13], implicating crosstalk of G-protein-coupled receptors (GPCRs) such as GRP receptor with EGFR. Other small intracellular proteins, such as Ras and non-receptor tyrosine kinase Src, have also been implicated in the crosstalk between GPCR and EGFR and activation of mitogen-activated kinase in COS-7 cells [16].
In addition to the activation of MAPK, other important signaling pathways related to cell survival and proliferation could be initiated following GRP-induced transactivation of EGFR. Protein kinase B/Akt has been recently demonstrated to play a pivotal role in cell survival through the regulation of cell cycle progression and apoptosis [17], [18]. Activation of Akt by phosphorylation is critical for cancer cell proliferation and survival triggered by growth factors, cytokines and extracellular matrix proteins [19], [20]. Akt is constitutively active in some NSCLC cells and promotes their survival [21]. Akt phosphorylation status and Akt-mediated anti-apoptotic effects are predominant factors in the efficacy of gefitinib, a specific EGFR tyrosine kinase inhibitor used clinically for NSCLC treatment [22], [23], [24]. The effect of GRP on cell survival and the involvement of PI3K-Akt signaling pathways downstream of GRPR activation have not been thoroughly explored.
In the present study, we examined GRP-induced signaling pathways and investigated the effects of GRP on the viability of NSCLC cells exposed to gefitinib. We found that GRP induced Akt phosphorylation and activation through a Src-dependent extracellular release of amphiregulin, leading to activation of EGFR. The release of amphiregulin and Akt activation are associated with the protective effect of GRP on the survival of NSCLC cells exposed to gefitinib. The GRP/GRPR pathway may be an important factor in the clinically observed resistance of NSCLC to EGFR inhibitors.
Section snippets
Cell lines and culture conditions
NSCLC cell lines 201T, 273T, and 128-88T were previously established in our laboratory from primary tumor tissue specimens [6]. The 273T cell carries a point mutation of EGFR at Y727C [25]. The cells were maintained in Basal Medium Eagle (BME) (Invitrogen Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum. A549 cells (wild-type EGFR) were obtained from American Type Culture Collection (Rockville, MD) and maintained in BME supplemented with 5% fetal bovine serum. Cells were cultured in
NSCLC cells express GRPR
Expression of the GRPR gene was examined in different NSCLC cell lines using a quantitative RT-PCR assay. Because H345 is a SCLC cell line known to express a high level of GRPR, we measured the GRPR mRNA relative to H345 cells. Our data showed that most examined NSCLC cell lines express higher GRPR mRNA than human bronchial epithelial cells (NHBE, BEAS2B), although relatively lower than H345 cells. As shown in Fig. 1, the GRPR mRNA is 8-fold higher in bronchioalveolar A549 cells, and 4-fold
Discussion
In the current study we present evidence that GRP stimulates phosphorylation of Akt that is dependent on EGFR and c-Src, in association with decreased effectiveness of the EGFR inhibitor gefitinib, an effect that is at least partially mediated through release of amphiregulin. A monoclonal antibody against GRP has been shown to inhibit SCLC growth in a xenograft mouse model [8], and the role of GRP/GRPR has been documented in many other malignant tumors [5], [6], including squamous carcinoma
Acknowledgments
The authors thank Dr. Robert Schultz from NCI for kindly providing Akt inhibitor API-2. We also thank Drs. Tony Godfrey and Liqiang Xi for their technical assistance on the real-time RT-PCR assay, and Dr. Edwina C. Kinchington for her helpful discussions.
The study was supported by the National Cancer Institute Lung Cancer SPORE grant P50 CA90440 (J.M.S.).
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2020, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :Our results demonstrate both NRG-1 and Bn-transactivation stimulate each of these signaling cascades in each NSCLC cell-line, and they show both similarities and differences from results in other cells. Our results which show a rapid activation of ERK and Akt in each NSCLC cells with Bn-transactivation are similar to studies reporting endothelin-transactivation of EGFR in fibroblasts [70] and GRP-transactivation of the EGFR/HER-family in other lung cancer cells [42,44]. Similarly, our results demonstrating sustained activation of Akt with NRG-1 stimulation agree with results in breast cancer cells [23,75] and prostate cancer cells [76].
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Current address: Cell and Molecular Biology Graduate Program, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.