SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

doi:10.1016/j.lfs.2014.01.072

Life Sciences

Volume 100, Issue 1, 28 March 2014, Pages 25-34

https://doi.org/10.1016/j.lfs.2014.01.072 Get rights and content

Abstract

Aims

The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated.

Main methods

The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1.

Key findings

Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells.

Significance

These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism.

Introduction

Neurotensin (NTS) (Carraway and Leeman, 1973) has potent growth effects in normal and neoplastic tissues (Evers, 2006). NTS is medullary thyroid carcinoma (Zeytinoglu et al., 1995) and small cell lung cancer (SCLC) cells (Moody et al., 1985). NTS is secreted from SCLC cells and binds with high affinity (Moody et al., 2003). The action of NTS is mediated by NTSR1 and NTSR2 as well as NTSR3, which has a single transmembrane domain and binds sortolin with high affinity (Betancur et al., 1998). SR48692 is a non-peptide NTSR1 antagonist (Gulley et al., 1993) which inhibits the proliferation of pancreatic, prostate and SCLC cells in vitro and in vivo (Moody et al., 2001, Valerie et al., 2011, Wang et al., 2011).

NTSR1 activation causes phosphatidylinositol (PI) turnover in a phospholipase C dependent manner (Dupouy et al., 2011). The inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) released elevation of cytosolic Ca^2 + (Staley et al., 1989) and activate protein kinase (PK)C, respectively (Muller et al., 2011). The activation of ERK and PKD is dependent upon PKC activity (Guha et al., 2002, Kisfalvi et al., 2005). NTS activates Akt and NF-κB pathways leading to increased cellular survival (Hassan et al., 2004, Zhao et al., 2003) and inactivates glycogen synthase kinase leading to increased cyclin D1 expression (Wang et al., 2006). NTS causes tyrosine phosphorylation of focal adhesion kinase (FAK) (Leyton et al., 2002) and Src (Lee et al., 2001). NTS causes epidermal growth factor (EGF)R and ERK tyrosine phosphorylation in prostate cancer cells (Hassan et al., 2004). The results indicate that NTS causes tyrosine phosphorylation of numerous proteins (Servotte et al., 2006, Heakal et al., 2011).

The NTSR1 is present in several types of cancer. Reubi et al. (1999) found a high density of specific (¹²⁵I–Tyr³)NTS binding sites in Ewing's sarcoma and medullary thyroid cancers. In non-small cell lung cancer (NSCLC), NTS and NTSR1 immunoreactivity are present in approximately 60% of lung adenocarcinoma biopsy specimens (Alfano et al., 2010). Patients with high NTSR1 had significantly lowers relapse-free survival than those with reduced NTSR1 levels. Similarly, high NTSR1 expression is associated with poor prognosis of patients with ductal breast cancer as well as head and neck squamous carcinomas (Dupouy et al., 2009, Shimizu et al., 2008). Treatment of mice containing NSCLC or colon cancer xenografts with the NTSR1 antagonist SR48692 reduced tumor growth (Moody et al., 2001, Maoret et al., 1999). These results suggest that NTSR1 may regulate the proliferation of numerous cancers.

The mechanism by which SR48692 inhibits NSCLC proliferation was investigated. Addition of siRNA to the NSCLC cells decreased significantly NTSR1 protein and decreased NTS transactivation of the EGFR and the ability of SR48692 to inhibit proliferation. The ability of NTS to cause EGFR tyrosine phosphorylation was inhibited by SR48692, gefitinib (EGFR TKI), GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) and U73122 (phospholipase C inhibitor). NTS stimulated, but gefitinib or SR48692 inhibited the clonal growth of NCI-H1299 cells. These results indicate that SR48692 inhibits the growth of NSCLC cells in an EGFR dependent mechanism.

Section snippets

Cell culture

NSCLC NCI-H1299 or A549 cells, which contain NTSR1 and wild type EGFR, were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY). The cells were split weekly 1/20 with trypsin-ethylenediaminotetraacetic acid (EDTA). The cells were mycoplasma-free and were used when they were in exponential growth phase after incubation at 37 °C in 5% CO₂/95% air.

Receptor binding

NCI-H1299 cells were plated in 24 well plates. When the

NTSR1 knock-down and effects of SR48692 on NSCLC cells

NTSR1 was investigated in NSCLC cells by Western blot and receptor binding. Table 1 shows that specific ¹²⁵I–Tyr³-NTS binding to NCI-H1299 cells was inhibited with high affinity by NTS, NTS^8–13, Ac-NTS^8–13 but not NTS^1–8 with IC₅₀ values of 4, 10, 7 and > 2000 nM, respectively. Also, SR48692 inhibited specific binding of radiolabelled NTS to NCI-H1299 cells (IC₅₀ value of 205 nM), whereas levocabastine, a NTSR2 agonist, and gefitinib did not (IC₅₀ values of > 2000 and > 2000 nM, respectively). Table 1

Discussion

NSCLC patients are treated traditionally with combination chemotherapy; however, the 5 year patient survival rate is only 16%. EGFR tyrosine kinase inhibitors have been approved for treating NSCLC patients who fail chemotherapy (Lynch et al., 2004, Paez et al., 2004). EGFR mutations of the ATP binding site are associated with patient response to EGFR tyrosine kinase inhibitors (Helfrich et al., 2006). Approximately 88% of the NSCLC patients, however, have wild type EGFR with reduced sensitivity

Conclusion

SR48692, a NTSR1 antagonist, decreases the proliferation of NSCLC cells in an EGFR-dependent manner. Knock-down of the NTSR1 decreases the growth inhibitory and EGFR transactivation effects of SR48692 on NSCLC cells. Using untreated NSCLC cells, the ability of NTS to increase EGFR transactivation is inhibited by gefitinib or SR48692. The proliferation of NSCLC cells is inhibited by gefitinib or SR48692 in vitro, however their combination has enhanced activity. It remains to be determined if the

Conflicts of interest statement

The authors declare that there is no conflict of interest.

Acknowledgments

The authors thank Dr. David Wink for helpful discussions. This research is partially supported by the intramural research program of the NIDDK and NCI, of NIH.

References (41)

R. Carraway et al.
The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalamus
J Biol Chem
(1973)
R. Cianfrocca et al.
The endothelin A receptor and epidermal growth factor receptor signaling converge on β-catenin to promote ovarian cancer metastasis
Life Sci
(2012)
S. Dupouy et al.
The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers
Biochimie
(2011)
L. Faivre et al.
A simple HPLC-UV method for the simultaneous quantification of gefitinib and erlotinib in human plasma
J Chromatogr B
(2011)
J.E. Hall et al.
Focal adhesion kinase: exploring FAK structure to gain insight into function
Int Rev Cell Mol Biol
(2011)
S. Hassan et al.
Involvement of MAP kinase, PI-3 kinase and EGF-receptor in the stimulatory effect of neurotensin on DNA synthesis in PC3 cells
Regul Pept
(2004)
R.T. Jensen et al.
Bombesin-related peptides and neurotensin: effects on cancer growth/proliferation and cellular signaling in cancer
J. Leyton et al.
Neurotensin causes tyrosine phosphorylation of focal adhesion kinase in lung cancer cells
Eur J Pharmacol
(2002)
T.W. Moody et al.
Neurotensin is produced by and secreted from classic small cell lung cancer cells
Life Sci
(1985)
T.W. Moody et al.
SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells
Peptides
(2001)

T.W. Moody et al.

Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells

Eur J Pharmacol

(2010)

J. Staley et al.

Neurotensin elevates cytosolic calcium in small cell lung cancer cells

Peptides

(1989)

Q. Wang et al.

Neurotensin phosphorylates GSK-3alpha/beta through the activation of PKC in human colon cancer cells

Neoplasia

(2006)

J.I. Wang et al.

Pancreatic cancer bears overexpression of neurotensin and neurotensin receptor subtype-1 and SR48692 counteracts neurotensin induced cell proliferation in human pancreatic ductal carcinoma cell line PANC-1

Neuropeptides

(2011)

S. Zhang et al.

Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer

Lung Cancer

(2008)

M. Alfano et al.

Neurotensin receptor 1 determines the outcome of non-small cell lung cancer

Clin Cancer Res

(2010)

G.P. Amorino et al.

Neurotensin stimulates mitogenesis of prostate cancer cells through a novel c-Src/Stat5b pathway

Oncogene

(2007)

C. Betancur et al.

Nonpeptide antagonists of neuropeptide receptors: tools for research and therapy

TIPS

(1998)

C. Cheng-Hsien et al.

Src homology 2-containing phosphotyrosine phosphatase regulates endothelin 1-induced epidermal growth factor receptor transactivation in rat renal tubular cell NRK-52E

Pflugers Arch

(2006)

S. Dupouy et al.

The neurotensin receptor-1 pathway contributes to human ductal breast cancer progression

PLoS ONE

(2009)

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Neurotensin receptor 1 (55 kDa) was present in NCI–H838, H358, H1299, H157 and A549 cells. Neurotensin receptor 1 was biologically active in that seconds after addition of 100 nM NTS to NCI–H1299 or A549 cells, the cytosolic Ca2+ was elevated (Moody et al., 2014). Also, Fig. 2 shows that receptor tyrosine kinases are present in NSCLC cells.
Neurotensin is a 13 amino acid peptide which is present in many lung cancer cell lines. Neurotensin binds with high affinity to the neurotensin receptor 1, and functions as an autocrine growth factor in lung cancer cells. Neurotensin increases tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and the neurotensin receptor 1 antagonist SR48692 blocks the transactivation of the EGFR. Here the effects of reactive oxygen species on the transactivation of the EGFR and HER2 were investigated. Using non-small cell lung cancer (NSCLC) cell lines, neurotensin receptor 1 mRNA and protein were present. Using NCI–H838 cells, neurotensin or neurotensin⁸⁻¹³ but not neurotensin¹⁻⁸ increased EGFR, ERK and HER2 tyrosine phosphorylation which was blocked by SR48692. Neurotensin addition to NCI–H838 cells increased significantly reactive oxygen species which was inhibited by SR48692, Tiron (superoxide scavenger) and diphenylene iodonium (DPI inhibits the ability of NADPH oxidase and dual oxidase enzymes to produce reactive oxygen species). Tiron or DPI impaired the ability of neurotensin to increase EGFR, ERK and HER2 tyrosine phosphorylation. Neurotensin stimulated NSCLC cellular proliferation whereas the growth was inhibited by SR48692, DPI or lapatinib (lapatinib is tyrosine kinase inhibitor of the EGFR and HER2). Lapatinib inhibited the ability of the neurotensin receptor 1 to transactivate the EGFR and HER2. The results indicate that neurotensin receptor 1 regulates the transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner.
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The combination of gefitinib and SR48692 showed an enhanced activity in inhibiting cell growth in vitro. It remains to be determined if the use of SR48692 increases the potency of gefitinib in NSCLC cells in vivo.69 Similarly, it has been shown that a combination of the BKR antagonist CU201 and gefitinib increased antitumor effects in head and neck cancer cells (HNSCC) in comparison to the effects of the single agents alone.50
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Published by Elsevier Inc.

SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

Abstract

Aims

Main methods

Key findings

Significance

Introduction

Section snippets

Cell culture

Receptor binding

NTSR1 knock-down and effects of SR48692 on NSCLC cells

Discussion

Conclusion

Conflicts of interest statement

Acknowledgments

J Biol Chem

Life Sci

Biochimie

J Chromatogr B

Int Rev Cell Mol Biol

Regul Pept

Eur J Pharmacol

Life Sci

Peptides

Eur J Pharmacol

Peptides

Neoplasia

Neuropeptides

Lung Cancer

Neurotensin receptor 1 determines the outcome of non-small cell lung cancer

Clin Cancer Res

Neurotensin stimulates mitogenesis of prostate cancer cells through a novel c-Src/Stat5b pathway

Oncogene

Nonpeptide antagonists of neuropeptide receptors: tools for research and therapy

TIPS

Src homology 2-containing phosphotyrosine phosphatase regulates endothelin 1-induced epidermal growth factor receptor transactivation in rat renal tubular cell NRK-52E

Pflugers Arch

The neurotensin receptor-1 pathway contributes to human ductal breast cancer progression

PLoS ONE