Widespread metastases are characteristic of the most aggressive form of lung
cancer, small cell lung cancer (SCLC). Although initially sensitive to treatment by
radio- and chemotherapy, SCLC develops chemoresistance so the 2-year survival
rate remains less than 5%. The aberrant proliferation of SCLC is sustained by
multiple autocrine and paracrine growth loops involving calcium mobilising
neuropeptides such as vasopressin (AVP) and gastrin releasing peptide (GRP). The
expression of these neuropeptides and their receptors are a hallmark of the disease
and present an important target for therapeutic intervention. Analogues of
substance-P, including [D-ArgI,D-Phe5,D-Trp7-9,Leu11]-substance-P (SP-D) and
[Arg6,D-Trp7-9,NmePhe8]-substance-P (6-11) (SP-G), are novel anti-cancer agents
which inhibit the growth of SCLC cells. Investigations into the mechanism of action
of substance- P analogues on bombesin receptors revealed that in addition to
blocking bombesin-induced mitogenesis and signal transduction they also have
agonist activity. This unique pharmacological activity of 'biased agonism' may be
centred to the growth inhibitory effects of these agents.
The aim of this study was to determine whether these agents exhibit 'biased agonist'
activity at receptors other than the bombesin/GRP receptor and investigate factors
influencing their ability to modulate neuropeptide signalling. Model cell systems
consisting of CHO-K1 cell-lines stably expressing GRP or Via receptors were
therefore established and the effect of SP-D and SP-G tested. Expression of GRP and
Via receptors led to the development of a transformed phenotype as cells showed
increased cloning efficiency and survival in soft-agar and suspension growth
respectively. GRP and Via receptor expressing cells were less adherent, more
migratory and not contact inhibited. Neuropeptide receptor stimulation provided
some protection from the cytotoxic effects of etoposide suggesting a role in
chemoresistance. Substance-P analogues inhibited normal and anchorageindependent
growth of receptor expressing cells. In receptor binding studies on
GRP and Via receptor expressing cells, analogue inhibited radioligand binding noncompetitively.
Transfected GRP and Via receptors effectively coupled to Ga.q to
increase intracellular calcium and the analogues were effective antagonists of this
response. Neuropeptide and analogues stimulated ERK activity in GRP and Via
receptor expressing cells. Activation of ERK by neuropeptide was rapid and
transient while analogue induced activation was delayed and sustained. Analoguestimulated
ERK activity was pertussis toxin sensitive whereas neuropeptidestimulated
ERK activation was not. In addition, analogue induced ERK activity was
blocked by inhibition of EGF receptor kinase. This indicates that SP-D and SP-G
facilitate receptor coupling to G-protein Gj/Gq subunits for subsequent calciumindependent
ERK activation via EGFR transactivation. Stable cell-lines expressing
different levels of Via receptor were used to examine the effect of altering the ratio
of receptor to G-protein on the ability of the analogues to direct receptor signalling.
There appeared to be little receptor reserve for calcium and ERK responses
stimulated by neuropeptide as the efficacy of the response increased with increasing
receptor numbers. In contrast, analogue-induced ERK activation occurred with a
higher receptor reserve for activated Gj as the magnitude of the response did not
increase between medium and high expressing cells. Chimeric Via receptors
containing the second (Vjj2) or third intracellular (Vii3) loop of the V2 receptor were
used to investigate the influence of substance-P analogues on G-protein selectivity.
Both receptors were still capable of binding AVP and SP-G but had altered ability to
activate PLC and ERK. The second intracellular loop of VIAR was essential for
AVP-stimulated PLC and ERK activation but not for SP-G-induced ERK activation.
This confirms that the effects of the agents cause an alteration in the receptor-Gprotein
coupling domains of receptors. These findings demonstrate that substance-P
analogues are biased agonists of receptors other than GRP receptors, activating
downstream signals which differ from those stimulated by the natural agonist
through promoting an alternative agonist state of the receptor. This pathway
selectivity combined with the receptor specificity of different substance-P analogues
offers great potential for the tailored treatment of neuropeptide-dependent tumours.