Neuropeptide S receptor 1 (NPSR1) activates cancer-related pathways and is widely expressed in neuroendocrine tumors

Abstract

Neuroendocrine tumors (NETs) arise from disseminated neuroendocrine cells and express general and specific neuroendocrine markers. Neuropeptide S receptor 1 (NPSR1) is expressed in neuroendocrine cells and its ligand neuropeptide S (NPS) affects cell proliferation. Our aim was to study whether NPS/NPSR1 could be used as a biomarker for neuroendocrine neoplasms and to identify the gene pathways affected by NPS/NPSR1. We collected a cohort of NETs comprised of 91 samples from endocrine glands, digestive tract, skin, and lung. Tumor type was validated by immunostaining of chromogranin-A and synaptophysin expression and tumor grade was analyzed by Ki-67 proliferation index. NPS and NPSR1 expression was quantified by immunohistochemistry using polyclonal antibodies against NPS and monoclonal antibodies against the amino-terminus and carboxy-terminus of NPSR1 isoform A (NPSR1-A). The effects of NPS on downstream signaling were studied in a human SH-SY5Y neuroblastoma cell line which overexpresses NPSR1-A and is of neuroendocrine origin. NPSR1 and NPS were expressed in most NET tissues, with the exception of adrenal pheochromocytomas in which NPS/NPSR1 immunoreactivity was very low. Transcriptome analysis of NPSR1-A overexpressing cells revealed that mitogen-activated protein kinase (MAPK) pathways, circadian activity, focal adhesion, transforming growth factor beta, and cytokine-cytokine interactions were the most altered gene pathways after NPS stimulation. Our results show that NETs are a source of NPS and NPSR1, and that NPS affects cancer-related pathways.

Fig. 1

Epitope specificities of the monoclonal anti-NPSR1-A and anti-NPSR1-N (detecting both NPSR1-A and NPSR1-B isoforms) antibodies against recombinant NPSR1 proteins overexpressed in COS-7 cells. The cell nuclei were stained with 40,6-diamino-2-phenylindole (DAPI) (left panel). Cells were transfected with the full-length variant for NPSR1-A fused with red fluorescence protein (NPSR1-A-pDsRed) or with the full-length variant for NPSR1-B green fluorescent protein (NPSR1-B-GFP) for 24 h and visualized under conventional fluorescence microscope (left middle). Cells transfected with NPSR1-A-pDsRed were stained either with the monoclonal anti-NPSR1-A or anti-NPSR1-N antibodies followed with secondary anti-mouse antibodies labeled with green (FITC) fluorescent tags, and cells transfected with NPSR1-B-GFP were stained with the monoclonal anti-NPSR1-N followed with secondary anti-mouse antibodies labeled with red fluorescent tags (TRITC) (right middle). The cells were permeabilized for staining with the anti-NPSR1-A antibodies, whereas the cells for anti-NPSR1-N stainings were left untreated. Overlay of the two images (right panel)

Fig. 2

Expression of NPSR1-N, NPSR1-A, and NPS in NETs and colon adenocarcinomas (mean ± SEM)

Fig. 3

Representative photomicrographs of NPSR1-N (a–c), NPSR1-A (d–f), and NPS (g–i) in NETs from pancreas (a, d, g), pheochromocytoma (b, e, h), and paraganglioma (c, f, i). Original magnification × 600

Fig. 4

Time and dose course of the gene induction upon NPS treatment. Parental SH-SY5Y cells and cells stably transfected with the NPSR1-A-GFP construct were stimulated a with NPS 100 nM for 3–24 h or b with 0.001–1 μM NPS for 3 h. mRNA levels of growth arrest and DNA-damage-inducible, alpha (GADD45A), nuclear receptor subfamily 4, group A, member 1 (NR4A1), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1), and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were measured by quantitative RT-PCR. The results are shown as average of three biological replicates (time) or three technical replicates (dose) (mean ± SD). The relative change in expression was calculated by comparing to the expression levels in unstimulated cells