NeuroD1 mediates nicotine-induced migration and invasion via regulation of the nicotinic acetylcholine receptor subunits in a subset of neural and neuroendocrine carcinomas

Abstract

Cigarette smoking is a major risk factor for acquisition of small cell lung cancer (SCLC). A role has been demonstrated for the basic helix-loop-helix transcription factor NeuroD1 in the pathogenesis of neural and neuroendocrine lung cancer, including SCLC. In the present study we investigate the possible function of NeuroD1 in established tumors, as well as actions early on in pathogenesis, in response to nicotine. We demonstrate that nicotine up-regulates NeuroD1 in immortalized normal bronchial epithelial cells and a subset of undifferentiated carcinomas. Increased expression of NeuroD1 subsequently leads to regulation of expression and function of the nicotinic acetylcholine receptor subunit cluster of α3, α5, and β4. In addition, we find that coordinated expression of these subunits by NeuroD1 leads to enhanced nicotine-induced migration and invasion, likely through changes in intracellular calcium. These findings suggest that aspects of the pathogenesis of neural and neuroendocrine lung cancers may be affected by a nicotine- and NeuroD1-induced positive feedback loop.

FIGURE 1:

Effect of nicotine on expression of NeuroD1 in patient-derived lung cancer, bronchial epithelial, and neuroendocrine cell lines. (A) Patient-derived SCLC, HBEC3KT, Clone 5, SHSY5Y, NTERA2, and rodent PC12 cell lines were lysed, and 25 μg of total protein was resolved on gels and immunoblotted for NeuroD1; GAPDH was the loading control. (B) Immortalized bronchial epithelial cells HBEC3KT, HBEC4KT, and HBEC30KT were treated with 0.25, 0.5, 1, and 2 μM nicotine for 24–48 h. Cells were fixed and immunostained for NeuroD1 and 4′,6-diamidino-2-phenylindole (DAPI). (C) qRT-PCR analysis of NeuroD1 expression in HBEC3KT, Clone5, HBEC30KT, and NSCLC HCC4017 with or without exposure to 0.25 μM nicotine for 48 h, plotted as relative expression. The control was 18s RNA. One of two independent experiments in duplicate. (D) HBEC3KT and Clone 5 cells were treated with 1 μM nicotine for 48 h, and 25 μg of lysate protein was immunoblotted as in A with tubulin as control. Relative expression quantified using LI-COR infrared imaging in three independent experiments; *p <0.05.

FIGURE 2:

Effect of nicotine and common signaling inhibitors on NeuroD1 expression and colony formation in lung cancer and immortalized bronchial epithelial cells. (A) Differential interference contrast images at 10× magnification of SCLC and NSCLC-neuroendocrine (NE) lines. (B) ERK1/2 and pERK1/2 were immunoblotted in 25 μg of lysate protein from HBEC, SCLC, and NSCLC cells as in Figure 1. (C) Formation of colonies in soft agar by SCLC (H69, H82) and NSCLC (H358, H460) cells after 4-wk exposure to 0, 0.1, 1, and 10 μM PD0325901. Colony number plotted as percentage of control. (D) SCLC H82, H69, and H2171 lines were starved for 18 h in 0.1% FBS and then untreated (NT) or treated with 1 μM nicotine for 15 and 120 min. ERK1/2 and pERK1/2 were immunoblotted as in B. (E) HBEC30KT and HCC4017 cells in normal medium were treated for 2 h as follows: A, control; B–E, 1 μM nicotine; C, +0.5 μM PD0325901; D, +10 μM LY294002; E, +10 μM mecamylamine. Lysates were immunoblotted for NeuroD1 and ERK1/2 as before. One of three independent experiments.

FIGURE 3:

Nicotine can increase NeuroD1 expression without causing neuroendocrine differentiation. (A, B) NTERA2 and SHSY5Y cells were treated with 25 μM nicotine or 25 μM retinoic acid for 14 d to induce differentiation. (A) Cells were fixed and immunostained for NeuroD1 and NeuN (mature neuronal marker) and DAPI. Representative of three independent experiments. (B) Cell lysates were immunoblotted for NeuroD1, TrkB, ASCL1, NGN3, and p53. GAPDH was the loading control. (C) HBEC3KT cells were treated with 0.63, 1.25, 2.5, and 5 μM nicotine over 24–48 h. Cell lysates were immunoblotted for p53; GAPDH was the loading control.

FIGURE 4:

NeuroD1 regulates the expression of α3 and α5, but not β4, nAChR subunits in SHSY5Y, Clone 5, SCLC, and NSCLC-NE lines. (A) NeuroD1 and α3, α5, and β4 subunits were immunoblotted in 25 μg of lysate protein from SCLC, NSCLC, HBEC3KT, Clone 5, and SHSY5Y cells as in Figure 1A. (B) SHSY5Y was treated with 0.1, 0.5, or 2.5 μM nicotine for 48 h. qRT-PCR of NeuroD1 and CHRNA3, A5, and B4 subunits; 18s RNA control. One of three independent experiments. *** p < 0.001, **p < 0.005. (C, D) Knockdown of NeuroD1 in H82, H1155, and Clone 5 lung and SHSY5Y neuroblastoma lines. H82 and H1155 cells were stably infected with shNeuroD1 or shControl vectors. Cells were selected by puromycin resistance and sorted for expression of green fluorescent protein. Then (C) NeuroD1 and CHRNA3, A5, A7, and B4 expression was analyzed by qRT-PCR and plotted relative to 18s RNA, or (D) lysate proteins were immunoblotted for NeuroD1 and α3, α5, and β4 with GAPDH as control.

FIGURE 5:

Acetylcholine receptor subunits α3 and α5 contribute to invasion induced by nicotine in SHSY5Y and clone 5 cells. (A) SHSY5Y and (B) clone 5 cells were infected with shNeuroD1, shα3, shα5, or shControl vectors to deplete the targeted proteins. Cells were dissociated, and 25,000 cells were placed in 0.2 ml of growth factor–reduced Matrigel. Invasion was scored as described in Materials and Methods. SHSY5Y, N = 2; Clone 5, N = 3. *p <0.05, **p <0.005. Right, proteins remaining 48 h after treatment with shRNAs, with tubulin as the loading control.

FIGURE 6:

TrkB, a downstream target of NeuroD1, regulates expression of several nAChR subunits in H69 and H82 SCLC and H1155 NSCLC-NE lines. (A) H69, H82, and H1155 cells were stably infected with shTrkB or shControl vectors. Cells were selected by sorting as in Figure 5. qRT-PCR analysis of TrkB and CHRNA3, A5, A7, and B4, plotted as expression relative to the 18s RNA control. (B) H69 and H82 cells were treated with 10 μM lestaurtinib for 8 h, followed by qRT-PCR analysis of TrkB and CHRNA3, A5, A7, B4, and B2 plotted as described. (C) H82 cells were starved for 8 h in 0.1% FBS. Cells were treated with 1 μM nicotine and 50 ng/ml BDNF for 1 h with or without lestaurtinib. Left, cell lysates immunoblotted with indicated antibodies. Right, blots quantified using LI-COR infrared imaging.

FIGURE 7:

Depletion of NeuroD1 decreases nicotine-induced increases in intracellular free calcium in SHSY5Y cells. (A) The indicated cell lines were loaded with fura-2AM and then stimulated with 12.5 μM nicotine. Fluorescence was monitored every 0.74 s after stimulation. (B) SHSY5Y cells loaded with fura-2AM were stimulated with 12.5 μM nicotine plus mecamylamine, nifedipine, or both at 10 μM, and fluorescence was monitored as described. N = 2. (C) SHSY5Y cells were infected with shNeuroD1, shα3, shα5, or shControl vectors for 48 h and then stimulated with 12.5 μM nicotine. Fluorescence was monitored as described. Representative of (A) four, (B) two, and (C) three independent experiments. Bottom, immunoblots showing effectiveness of depletion.

FIGURE 8:

Model. Nicotine induces activation of NeuroD1 and the nAChR subunits. NeuroD1 then regulates its downstream targets, TrkB and the nAChR subunits, which control invasion and migration in cancer cells.