SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner
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 Ca2 + (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, NTS8–13, Ac-NTS8–13 but not NTS1–8 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.
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