Presynaptic neuronal antigens expressed by a small cell lung carcinoma cell line
Introduction
Small cell lung carcinomas (SCLC) are aggressive tumours, which show a strong association with cigarette smoking and represent approximately 25% of all lung carcinomas. The tumours proliferate rapidly, metastase early and although they may respond, at least initially, to chemotherapy the patient’s long-term survival is usually less than 10%. SCLCs are often associated with autoimmune neurological disorders such as the Lambert–Eaton myasthenic syndrome (LEMS), subacute sensory neuronopathy (SSN) or various forms of paraneoplastic encephalitis (PE) (Lang and Vincent, 1996). Sixty percent of LEMS patients have a SCLC, and greater than 90% of all LEMS patients have antibodies directed against P/Q-type VGCCs (Motomura et al., 1997). In patients with SSN or PE, the antibodies are often directed against a group of neuronal nuclear proteins called ‘Hu’, but patients with cerebellar ataxia and SCLC can have antibodies to VGCCs, Hu or both antigens (Mason et al., 1997).
SCLC cells exhibit a number of neurosecretory properties, including the expression of neurone-specific enolase and l-dopa decarboxylase, and the presence of autocrine factors such as bombesin and gastric-releasing factor. It has been suggested that cell proliferation is controlled by autocrine loops involving peptide secretion (Cuttitta et al., 1985, Carney, 1992) in a calcium-dependent manner (Sorenson et al., 1981). Cultured SCLC cells have been shown to express voltage-dependent calcium, potassium and sodium channels and to generate action potentials (McCann et al., 1981; Pancazio et al., 1992; Viglione et al., 1993, Blandino et al., 1995), and VGCCs have been identified both biochemically and electrophysiologically in a number of SCLC tumours and cell lines (De Aizpurua et al., 1988, Johansson et al., 1989, Oguru-Okano et al., 1992, Codignolo et al., 1993, Johnston et al., 1994; Sher et al., 1990). Depolarisation-induced calcium entry through VGCCs is coupled to neurotransmitter release in the neuroendocrine and nervous systems via multimolecular protein complexes comprising a combination of synaptic vesicle and plasma membrane proteins (Catterall, 1999). It is not known, however, whether the secretory machinery in SCLCs is subserved by the L- or N-type VGCC utilised in neuroendocrine systems, or by the P-/Q-type VGCCs that control release of neurotransmitter at the peripheral neuromuscular junction. In addition, recently a SCLC line has been shown to express both the muscle and the neuronal AChR (Sciamanna et al., 1997). Antibodies against neuronal AChR have been reported in patients with a number of different neurological disorders some of whom had an underlying SCLC (Vernino et al., 1998). Taken as a whole, these observations indicate that SCLC may express a wide range of neuronal antigens.
We derived a SCLC cell line (MB) from the tumour of a patient with LEMS and have shown the effect of LEMS IgG on this line (Johnston et al., 1994). Subsequently, this patient also developed antibodies to acetylcholine receptors (AChRs) suggesting that, surprisingly, they too might be expressed in her SCLC line. Here we have used molecular and functional studies to demonstrate the presence of many of the proteins involved in the excitation–secretion pathway in the nervous system and some of those expressed at the neuromuscular junction during development. The presence of a range of different neuronal/exocytotic antigens in these cells suggests that VGCCs and Hu antigens are probably not the only target antigens for paraneoplastic syndromes associated with SCLC. The presence of agrin and ARIA, as well as proteins involved in neurosecretory pathway, suggest that these cells display, surprisingly, a ‘motor nerve terminal’-like characteristic.
Section snippets
Cell cultures
MB cells were derived as previously described (Johnston et al., 1994), and were grown continuously as semi-adherent clusters in DMEM/F12 supplemented with 15 mM HEPES, 2% foetal calf serum (FCS), 2 mM l-glutamine, 50 units/ml penicillin, 50 units/ml streptomycin (all Gibco BRL) and Sato’s N1 components (Bottenstein and Sato, 1979) at 37°C. Fresh medium was added on day 5–6 and cultures were passaged every 10–12 days with trypsin/EDTA (Gibco/BRL). TE671 were grown in DMEM supplemented with 5%
Clinical details
M.B. was a moderate smoker who developed the clinical features of the Lambert–Eaton myasthenic syndrome (LEMS) at the age of 44 years. The diagnosis was confirmed by electromyography, revealing a reduced compound muscle action potential amplitude in the abductor digiti minimi (1.2 mV; normal values >8 mV). The amplitude increased by >100% following maximal voluntary activation. She was subsequently shown to be positive for anti-VGCC antibodies (Fig. 1). Initial computerised tomographic chest
Discussion
We have studied a SCLC cell line derived from a patient with LEMS who had a high titre of anti P-/Q-type VGCC antibodies. The cells expressed all of the components of the neurosecretory apparatus that were tested and transcripts for the subunits α1A, α2δ and the β subunits, which are thought to combine to encode a P-/Q-type VGCC. Expression of VGCCs of the P/Q-subtype was confirmed by radioligand binding, and specific antagonist inhibition of K+-induced 45Ca2+ flux. Moreover, it was this VGCC,
Acknowledgments
This work was supported by grants from the MRC, Muscular Dystrophy Association, Jules Thorn Trust and the Myasthenia Gravis Association.
M.B. was a Rhodes Scholar. F.B. was a recipient of a post-doctoral fellowship from the “Deutsche Forschunggsgemeinschaft” (Bl 452/1-1). The authors are very grateful to Professor John Newsom-Davis for providing the patients’ clinical information and to Ms. Eve Goodyer for the serum samples.
References (35)
Biology of small-cell lung cancer
Lancet
(1992)- et al.
Single-step method of RNA isolation by acid guanidine thiocyanate–phenol–chloroform extraction
Ann. Biochem.
(1987) - et al.
Expression of synaptotagmin and syntaxin associated with N-type calcium channels in small cell lung carcinoma
FEBS Lett.
(1993) - et al.
Autoimmunity to ion-channels and other proteins in paraneoplastic disorders
Curr. Opin. Immunol.
(1996) - et al.
Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/Heregulin, and its putative receptors ErbB2 and ErbB3, in developing mammalian muscle
Develop. Biol.
(1995) - et al.
Incidence of serum anti P-/Q-type and anti-N-type calcium channel autoantibodies in the Lambert–Eaton myasthenic syndrome
J. Neurol. Sci.
(1997) Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25
Gene
(1994)- et al.
Expression and antibody inhibition of P-type calcium channels in human small cell lung carcinoma cells
J. Neurosci.
(1995) - et al.
Voltage-dependent sodium channels in human small-cell lung cancer cells: role in action potentials and inhibition by Lambert–Eaton syndrome IgG
J. Membr. Biol.
(1995) - et al.
Growth of a rat neuroblastoma cell line in serum-free supplemented medium
Proc. Natl. Acad. Sci.
(1979)
The role of agrin in synapse formation
Ann. Rev. Neurosci
Nicotine stimulates a serotonergic autocrine loop in human small-cell lung carcinoma
Cancer Res.
Interactions of presynaptic Ca2+ channels and snare proteins in neurotransmitter release
Ann. N.Y. Acad. Sci.
Calcium channel subtypes controlling serotonin release from human small cell lung carcinoma cell lines
J. Biol. Chem.
Bombesin like peptides can function as autocrine growth factors in human small cell lung cancer
Nature
Antagonism of voltage-gated calcium channels in small cell carcinomas of patients with and without Lambert–Eaton myasthenic syndrome by autoantibodies, ω-conotoxin and adenosine
Cancer Res.
Identification of heregulin, a specific activator of p185erbB2
Science
Cited by (48)
Lambert-Eaton myasthenic syndrome
2024, Handbook of Clinical NeurologyAdvances in the understanding of disease mechanisms of autoimmune neuromuscular junction disorders
2022, The Lancet NeurologyCitation Excerpt :Several cellular and serological studies identified coexisting antibodies against other active zone proteins, and against CaV2.2 (N-type) VGCCs.65,66,77 In Lambert-Eaton myasthenic syndrome patients with small-cell lung cancer, these antibodies probably result from an immunological response to the small-cell lung cancer, expressing these antigens in addition to CaV2.1.78,79 However, such antibodies are thought to have a pathogenic role in only a minority of patients.77
Immunoglobulin Replacement Therapy
2022, Encyclopedia of Infection and ImmunityPulmonary manifestations of autoimmune diseases
2022, Translational Autoimmunity: Autoimmune Diseases in Different OrgansDiscordant SOX-1 antibodies results in paraneoplastic Lambert-Eaton syndrome diagnosis by the clinical laboratory
2021, Clinical BiochemistryCitation Excerpt :To confirm the diagnosis of LEMS, anti-voltage gated calcium channel (VGCC) antibodies were determined in an external laboratory, showing a considerably high concentration (369 pmol/L, Reference Values: 0–20 pmol/L). Expression of functional VGCCs in the surface membrane of small cell lung cancer (SCLC) cells (among numerous other neural antigens) likely account for most cases of paraneoplastic LEMS [1]. Lambert-Eaton myasthenic syndrome is a paraneoplastic or primary autoimmune neuromuscular junction disorder characterized by proximal weakness and autonomic dysfunction [2].
Neuronal antibody detection and improved lung cancer prediction in Lambert-Eaton myasthenic syndrome
2020, Journal of NeuroimmunologyCitation Excerpt :Half of the patients with GABAb antibodies (4/8) also had N-type VGCC antibodies, all with SCLC-LEMS. Both N-type VGCCs and GABAb are expressed in SCLC tissue (Benatar et al., 2001; Lancaster et al., 2010). Typically, patients with GABAb antibodies present almost exclusively with limbic encephalitis, in a paraneoplastic form in approximately 50% of cases, where the tumour is almost always found to be SCLC (Lancaster et al., 2010; Boronat et al., 2011; Höftberger et al., 2013; Jeffery et al., 2013; Chen et al., 2017; Dogan Onugoren et al., 2015; van Coevorden-Hameete et al., 2019).