Nicotinic acetylcholine receptors mediate lung cancer growth

Abstract

Ion channels modulate ion flux across cell membranes, activate signal transduction pathways, and influence cellular transport-vital biological functions that are inexorably linked to cellular processes that go awry during carcinogenesis. Indeed, deregulation of ion channel function has been implicated in cancer-related phenomena such as unrestrained cell proliferation and apoptotic evasion. As the prototype for ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) have been extensively studied in the context of neuronal cells but accumulating evidence also indicate a role for nAChRs in carcinogenesis. Recently, variants in the nAChR genes CHRNA3, CHRNA5, and CHRNB4 have been implicated in nicotine dependence and lung cancer susceptibility. Here, we silenced the expression of these three genes to investigate their function in lung cancer. We show that these genes are necessary for the viability of small cell lung carcinomas (SCLC), the most aggressive type of lung cancer. Furthermore, we show that nicotine promotes SCLC cell viability whereas an α3β4-selective antagonist, α-conotoxin AuIB, inhibits it. Our findings posit a mechanism whereby signaling via α3/α5/β4-containing nAChRs promotes lung carcinogenesis.

Keywords: CHRNA5; ligand-gated ion channel; lung cancer; nicotinic acetylcholine receptor; small cell lung carcinoma.

Figure 1

CHRNA3, CHRNA5, and CHRNB4 depletion decreases SCLC cell growth. DMS-53 cells were treated with three distinct CHRNA3, CHRNA5, and CHRNB4 siRNAs or a negative control siRNA (Applied Biosystems). (A) Quantitative RT-PCR was performed to determine mRNA levels upon knockdown. (B) A bioluminescence-based cell viability assay was performed to determine the effect of siRNA treatment on SCLC cell viability (^***p < 0.001, ANOVA, Tukey post-hoc test). (C) CHRNA5 mRNA levels in DMS-53 cells stably expressing a non-silencing shRNAmir or two CHRNA5 shRNAmirs (Open Biosystems). shRNAmirs were transduced into DMS-53 cells using a lentiviral delivery system for stable expression (Open Biosystems Trans-Lentiral Packaging System). (D) DMS-53 cells stably expressing a non-silencing shRNAmir or two CHRNA5 shRNAmirs were subcutaneously injected into the hind flanks of athymic nude mice. Tumors were harvested after 2 months (representative images shown). Tumor size (E) and weight (F) were significantly lower in samples treated with CHRNA5 shRNAmirs vs the non-silencing control (^*p < 0.05; ^**p < 0.01, ANOVA, Tukey post-hoc test).

Figure 2

Pharmacological activation or inhibition of nAChRs modulates SCLC growth. (A) DMS-53 cells were treated daily for 1 week with 1 μM nicotine or 2 μM α-conotoxin AuIB. Cell viability assays show that nicotine increases while AuIB decreases SCLC cell viability (values normalized to saline control; ^*p < 0.05, ^**p < 0.01). (B) DMS-53 cells were injected subcutaneously into the hind flanks of athymic nude mice. Mice were then implanted with osmotic minipumps that delivered either saline or 24 mg/kg of nicotine daily. Tumors were harvested after 1 month (representative images shown). (C,D). Chronic nicotine exposure increased both tumor size and weight (^**p < 0.01, Student's t-test).