Autocrine production of reproductive axis neuropeptides affects proliferation of canine osteosarcoma in vitro

Abstract

Background: Osteosarcoma strikes hundreds of people each year, of both advanced and younger ages, and is often terminal. Like many tumor types, these bone tumors will frequently undergo a neuroendocrine transition, utilizing autocrine and/or paracrine hormones as growth factors and/or promoters of angiogenesis to facilitate progression and metastasis. While many of these factors and their actions on tumor growth are characterized, some tumor-derived neuropeptides remain unexplored.

Methods: Using validated canine osteosarcoma cell lines in vitro, as well as cells derived from spontaneous tumors in dogs, we explored the autocrine production of two neuropeptides typically found in the hypothalamus, and most closely associated with reproduction: gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss-1). We evaluated gene expression and protein secretion of these hormones using quantitative RT-PCR and a sensitive radioimmunoassay, and explored changes in cell proliferation determined by MTS cell viability assays.

Results: Our current studies reveal that several canine osteosarcoma cell lines (COS, POS, HMPOS, D17, C4) synthesize and secrete GnRH and express the GnRH receptor, while COS and POS also express kiss1 and its cognate receptor. We have further found that GnRH and kisspeptin, exogenously applied to these tumor cells, exert significant effects on both gene expression and proliferation. Of particular interest, kisspeptin exposure stimulated GnRH secretion from COS, similarly to the functional relationship observed within the neuroendocrine reproductive axis. Additionally, GnRH and kisspeptin treatment both increased COS proliferation, which additionally manifested in increased expression of the bone remodeling ligand rankl within these cells. These effects were blocked by treatment with a specific GnRH receptor inhibitor. Both neuropeptides were found to increase expression of the specific serotonin (5HT) receptor htr2a, the activation of which has previously been associated with cellular proliferation, suggesting that production of these factors by osteosarcoma cells may act to sensitize tumors to circulating 5HT of local and/or enteric origin.

Conclusions: Here we report that kisspeptin and GnRH act as autocrine growth factors in canine osteosarcoma cells in vitro, modulating RANKL and serotonin receptor expression in a manner consistent with pro-proliferative effects. Pharmacological targeting of these hormones may represent new avenues of osteosarcoma treatment.

Keywords: Autocrine; GnRH; Kisspeptin; Osteosarcoma; Proliferation.

Fig. 1

(a) gnrh (b) kiss1, (c) gnrhr, and (d) gpr54/kiss1r mRNA expression in normal canine osteoblasts (OPC), multiple osteosarcoma cell lines (COS, POS, HMPOS, C4, D17, human SAOS) and clinical patient-derived samples (1014, 1036). Arrow in (b) indicates kiss1 band. PCR primers used can be found in Additional file 2 Table S1. e β-actin (actb) expression in OPC, COS, POS, HMPOS, and D17 cells. f Relative levels of cKiss1R expression in COS vs. OPC. g Immunoblot of Kiss1R protein (54kD, upper band) in normal canine osteoblasts (OPC) and osteosarcoma lines (COS, POS); α-tubulin (42kD, lower band) was also probed as a control. h RT-PCR expression of rank in COS vs. OPC. i RT-PCR expression of rankl in COS vs. OPC (3 preparations). NTC indicates RNA (no cDNA template) control

Fig. 2

a GnRH secretion in static incubation from media of COS in vitro treated for 4 h with 17β-estradiol (100.0 pM), kisspeptin-10 (10.0 nM), or a combination of E2 and kisspeptin-10. b Representative plot of GnRH secretion from perifused COS treated with either 0.05% DMSO vehicle or 10.0 nM kisspeptin-10. Lines indicate duration of exposure to drug/vehicle

Fig. 3

a Cell viability of COS following 24–48 h of treatment with 12.5 μM 5HT (2nd bar, *, p < 0.05; n = 6), 1.0 nM GnRH (3rd bar, *, p < 0.05; n = 6), 1.0 nM GnRH with 10.0 nM Teverelix (GnRHR inhibitor; 4th bar), or GnRH and 5HT co-treatment (last bar, **, p < 0.01; n = 6). b Cell viability of COS (as % of DMSO control) following increasing doses of either 5HT alone (grey line) or GnRH+5HT (black line). c Cell viability of COS following 24 h of treatment with 10 nM kisspeptin, 10 nM Teverelix, 10 nM each Kisspeptin+Teverelix, 1.0 nM GnRH, or 1.0 nM GnRH + 10 nM Teverelix (**, p < 0.01; n = 6; ***, p < 0.001; n = 6)

Fig. 4

a Changes in rank gene expression after treatment with 1.0 nM GnRH, 1.0 nM GnRH + 10.0 nM Teverelix, 10.0 nM kisspeptin, 100.0 pM 17β-estradiol, 1.0 nM GnRH + 100 pM 17β-estradiol, 10.0 nM kisspeptin + 100.0 pM 17β-estradiol, or 5HT (3.0 μM and 12.0 μM). b Changes in rankl gene expression after treatment with hormones described above (*, p < 0.05; **, p < 0.01, n = 6/treatment). c Changes in opg gene expression after treatment with hormones described above

Fig. 5

a Relative expression of htr2a (black bars) and htr1b (white bars) following 4 h treatment with 1.0 nM GnRH, 10.0 nM kisspeptin-10, or 100.0 pM 17β-E2. b Relative expression of htr2a and htr1b following 24 h (right panel) treatment with 1.0 nM GnRH, 10.0 nM kisspeptin-10, or 100.0 pM 17β-E2. (*, p < 0.05, n = 5 for each treatment group)

Fig. 6

a Representative plots of GnRH secretion from perifused COS treated with 12.5 μM 5HT for duration indicated by black bar. b GnRH secretion from perifused COS treated with 6.0 μM 5HTR2A-specific inhibitor ritanserin for duration indicated by grey bar