SMALL CELL LUNG CANCER CELLS: STIMULATION BY MULTIPLE NEUROPEPTIDES AND INHIBITION BY BROAD SPECTRUM ANTAGONISTS. A thesis submitted for the degree of Doctor of Philosophy in the University of London by TARIQJ. SETHI July 1993 Growth Regulation Laboratory Department of Cell Biology Imperial Cancer Research Fund University College London London ProQuest Number: 10017254 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10017254 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Human small cell lung cancer (SCLC) constitutes 25% of lung cancers and follows an aggressive clinical course. SCLC is characterised by the presence of intracytoplasmic neurosecretory granules and by its ability to secrete many hormones and neuropeptides. Only bombesin-like peptides, which include gastrin-releasing peptide (GRP), have been shown to act as autocrine growth factors for certain SCLC cell lines. This thesis focused on other neuropeptides and particularly their ability to mediate SCLC growth. The neuropeptides bradykinin, cholecystokinin (CCK), GRP, neurotensin and vasopressin at nanomolar concentrations stimulated an increase in the intracellular concentration of calcium ([Ca^+Jj), inositol phosphate hydrolysis, and increased colony formation in semi-solid medium in responsive SCLC cell lines. These results suggest that multiple Ca^+-mobilising neuropeptides stimulate SCLC growth by an extensive network of autocrine and paracrine interactions. With tumour progression these neuropeptides have increased potency. Galanin a 29 amino-acid peptide opposes Ca^+ signals and modulates the action of other neuropeptides in various cellular systems. Thus the effect of galanin in SCLC cell lines was investigated. Surprisingly, galanin increased rather than decreased [Ca2+]j, and stimulates the production of inositol phosphates in certain SCLC cell lines. In view of the Ca^+-mobilizing actions of galanin, the effect on SCLC growth was tested. Galanin stimulates clonal growth in SCLC cells, further supporting the proposition that SCLC growth is stimulated by multiple autocrine/paracrine interactions involving Ca^+ mobilizing neuropeptides. This is the first time that galanin is shown to evoke inositol phosphate, Ca^+ mobilisation and growth responses in any cell type. Gastrin, CCK and CCK-related peptides have identical carboxy-terminal amino-acid structure. SCLC cells are shown to express two distinct functional CCK receptor subtypes, CCK& and gastrin/CCKg receptors, both of which increase [Ca2+]j and stimulate clonal growth. The cDNA of the gastrin/CCKg receptor was cloned and sequenced. Northern blot analyses revealed a single transcript of 2.4 kb in SCLC. The levels of RNA expression match directly with the ability of these SCLC cells to respond to gastrin. This is the first time that CCK& and gastrin/CCKg receptors are shown to stimulate growth outside the gastointestinal system. The broad-spectrum neuropeptide antagonists [D-Arg** .D-Phe^, D- Trp^'^.Leu"! substance P and [Arg®,D-Trp^»®,MePhe®] substance P (6 - 11) block bradykinin, CCK, galanin, gastrin, GRP, neurotensin and vasopressin mediated signals and growth in SCLC cell lines, and inhibit SCLC growth in vitro and in vivo. Thus, broad spectrum neuropeptide antagonists constitute potential anticancer agents. CONTENTS CHAPTER 1 1.1 INTRODUCTION ...................................................................................................................1 1.2 CELL PROLIFERATION VIA THE G-PROTEIN PATHWAY.................................................... 3 1.2.a G protein-coupled receptors ................................................................................. 3 1.2.b G-proteins and phospholipase C ...........................................................................5 1.2.C Inositol Phosphates and Calcium ..........................................................................6 1.2.d Inositol phosphate metabolism and lithium .......................................................8 1.3. NEUROPEPTIDES AND CELL GROWTH................................................................................ 10 1.3.a Bombesin/Gastrin-releasing peptide ..................................................................10 1.3.b Bombesin/GRP as a growth factor ........................................ '.............................12 1.3.C Bombesin/GRP in development .............................................................................12 1.4. BOMBESIN/GRP SIGNAL TRANSDUCTION........................................................................... 13 1.4.a Bombesin/GRP Receptors ..................................................................................... 13 1.4.b Phosphatidyl inositol turnover, CaZ+ mobilisation and activation of protein kinase C..................................................................................................14 1.4.C Arachidonic acid release and prostaglandin synthesis: .................................. 16 1.4.d Bombesin induction of the proto-oncogenes c-fos and c-myc .................... 16 1.4.e Neuropeptide stimulation of tyrosine kinase activity................................... 17 1.4.f Regulation of cellular responsiveness to bombesin-stimulated .............. mitogenesis ...................................................................................................... 18 1.5. OTHER NEUROPEPTIDE GROWTH FACTORS...................................................................... 19 1.5.a Vasopressin ............................................................................................................... 19 1.5.b Bradykinin.................................................................................................................. 20 1.5.C Tachykinins................................................................................................................21 1 .S.d Vasoactive intestinal peptide ............................................................................... 22 1.6 . SIGNAL TRANSDUCTION BY TYROSINE KINASE RECEPTORS........................................ 23 1.6 .a Structural features of receptor tyrosine kinases.............................................23 1.6 .b Receptor activation .................................................................................................24 1.6.C Phosphatidylinositol 3-kinase ................................................................................25 1.6 .d Phospholipase C-y ...................................................................................................26 1.5.e GTPase-activating protein ....................................................................................27 1.6 .f SH2 domains mediate the interaction of signalling molecules to recep to rs .................................................................................................................. 28 1.7. POLYPEPTIDE GROWTH FACTORS AND CANCER............................................................. 28 1.8. LUNG CANCER .......................................................................................................................... 3 0 1.8.a INTRODUCTION .........................................................................................................30 1.8.b Classification of Lung Cancers..............................................................................31 1.8.C SMALL CELL LUNG CANCER: Clinical features.................................................32 1.9. Causative agents .....................................................................................................................3 5 1.10. GENETIC CHANGES.............................................................................................................. 35 1.10.a Hereditary predisposition - lung cancer families ........................................... 36 1.10.b Chromosomal abnormalities ............................................................................... 36 1.11. RECESSIVE ONCOGENES....................................................................................................37 1.11.a Retinoblastoma gene ............................................................................................37 1.1 l.b p53 .......................................................................................................................... 3 8 1.11.c 3p ............................................................................................................................. 3 9 1.1 l.d Other recessive oncogenes. ...............................................................................40 1.12. DOMINANT ACTING ONCOGENES....................................................................................40 1.12.a myc fam ily ............................................................................................................ .41 1.12.b ras family: K-, H- and N-ras. ........................................................................ 42 1.12.c Other dominant-acting oncogenes ............................................