Evidence for autocrine actions of neuromedin B and gastrin-releasing peptide in non-small cell lung cancer.
Gastrin-releasing peptide (GRP), a member of the bombesin family of peptides, has been shown to have mitogenic activity in small cell lung carcinoma (SCLC), and to be produced by SCLC in an autocrine fashion. In this report, we demonstrate that both GRP and another member of the bombesin family of peptides, neuromedin B (NMB), are also autocrine growth factors for non-small cell lung carcinoma (NSCLC). Using the reverse transcription-polymerase chain reaction (RT-PCR), we have detected mRNA for the neuromedin B receptor (NMBR) in all 14 of the NSCLC cell lines examined. GRP receptor (GRPR) mRNA was also expressed in the majority of NSCLC cell lines (nine of 14). By immunoblotting using SDS-PAGE gradient gels fixed in trichloroacetic acid, GRP and NMB were found in fractions of culture medium that had been purified by high pressure liquid chromatography (HPLC) from NSCLC cell lines. NMB was detected in the conditioned medium of seven of nine cell lines and GRP in seven of nine cell lines; both peptides were produced in six cell lines. In four of the cell lines where both peptides were produced, the relative amount of NMB secreted into the medium was 7-15 times that of GRP; in the other two cases, the relative amounts of GRP and NMB were equivalent. Cultured human bronchial epithelial (HBE) cells expressed the GRPR and NMBR but did not produce either peptide. A subline of A549 cells that was adapted to grow in serum-free and growth factor-free conditions, termed A549-R(0), secreted both bombesin-like peptides (BLPs) into the culture medium. Using either a colony-forming assay or a BrDU incorporation assay, both NMB and GRP were found to be mitogens for three NSCLC cell lines that express mRNA for BLP receptors and secrete BLPs, regardless of which peptide and/or receptor subtype was detected. The monoclonal antibody 2A11, which preferentially recognizes GRP, was able to block the in vitro proliferative response to GRP in the BrDU incorporation assay, and partially blocked the response to NMB. The 2A11 antibody could only partially block the in vivo growth of cell lines that showed proliferative responses to BLPs. 2A11 antibody was more effective against the 239T cell line, which secreted a low amount of GRP into the medium (0.6 nM), compared to the 201T cell line, which secreted a higher amount of both GRP and NMB (4.2 nM and 36.6 nM, respectively). These results suggest that both NMB and GRP are autocrine growth factors for NSCLC, but that the production of NMB and expression of the NMBR may be more prominent than the production of GRP and expression of the GRP receptor. If BLP ligand-receptor systems are to be targeted therapeutically in NSCLC, it will be necessary to inhibit both NMB and GRP.
Siegfried
J M
JM
Department of Pharmacology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, 15261, USA.
Krishnamachary
N
N
Gaither Davis
A
A
Gubish
C
C
Hunt
J D
JD
Shriver
S P
SP
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019898:Autocrine Communication / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D017077:Culture Media, Conditioned; D005260:Female; D019886:Gastrin-Releasing Peptide / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D008934:Mitogens / Q000494:pharmacology; D015287:Neurokinin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D018004:Receptors, Bombesin / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D008934:Mitogens; D012333:RNA, Messenger; D018004:Receptors, Bombesin; D019886:Gastrin-Releasing Peptide; D015287:Neurokinin B; C038393:neuromedin B