Oncostatin M promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines

Abstract

Background: We have previously demonstrated that both canine and human OSA cell lines, as well as 8 fresh canine OSA tumor samples, exhibit constitutive phosphorylation of STAT3, and that this correlates with enhanced expression of matrix metalloproteinase-2 (MMP2). While multiple signal transduction pathways can result in phosphorylation of STAT3, stimulation of the cytokine receptor gp130 through either IL-6 or Oncostatin M (OSM) is the most common mechanism through which STAT3 is activated. The purpose of this study was to evaluate the role of IL-6 and OSM stimulation on both canine and human OSA cell lines to begin to determine the role of these cytokines in the biology of OSA.

Methods: RT-PCR and Western blotting were used to interrogate the consequences of OSM and IL-6 stimulation of OSA cell lines. OSA cells were stimulated with OSM and/or hepatocyte growth factor (HGF) and the effects on MMP2 activity (gel zymography), proliferation (CyQUANT), invasion (Matrigel transwell assay), and VEGF production (Western blotting, ELISA) were assessed. The small molecule STAT3 inhibitor LLL3 was used to investigate the impact of STAT3 inhibition following OSM stimulation of OSA cells.

Results: Our data demonstrate that the OSM receptor (OSMR), but not IL-6 or its receptor, is expressed by all human and canine OSA cell lines and canine OSA tumor samples; additionally, OSM expression was noted in all tumor samples. Treatment of OSA cell lines with OSM induced phosphorylation of STAT3, Src, and JAK2. OSM stimulation also resulted in a dose dependent increase in MMP2 activity and VEGF expression that was markedly reduced following treatment with the small molecule STAT3 inhibitor LLL3. Lastly, OSM stimulation of OSA cell lines enhanced invasion through Matrigel, particularly in the presence of rhHGF. In contrast, both OSM and HGF stimulation of OSA cell lines did not alter their proliferative capacity.

Conclusions: These data indicate OSM stimulation of human and canine OSA cells induces STAT3 activation, thereby enhancing the expression/activation of MMP2 and VEGF, ultimately promoting invasive behavior and tumor angiogenesis. As such, OSM and its receptor may represent a novel target for therapeutic intervention in OSA.

Figure 1

Expression of OSM and IL-6 receptors in canine and human OSA. RNA was collected from untreated A) canine (OSA8, 16, and D17) or B) human (SJSA and U2OS) OSA cell lines and RT-PCR was performed for IL-6, IL-6 receptor, OSM, OSMR, gp130, and GAPDH. C) RNA was collected from eight fresh frozen OSA tumors from canine patients and normal canine osteoblasts (Nml K9 Ob) and RT-PCR performed for OSM, OSMR, and GAPDH.

Figure 2

Activation of STAT3, JAK2, and Src in OSA cell lines upon following stimulation with OSM. Canine (OSA8) or human (SJSA) OSA cell lines were serum starved for two hours then stimulated with rhOSM (50 ng/mL) for 0 (no OSM stimulation), 5, 10, or 30 minutes. Protein lysates were generated and separated by SDS-PAGE and Western blotting for pSTAT3 (Y705), total STAT3, pJAK2 (Y1007/1008), total JAK2, pSrc (Y418), and total Src was performed.

Figure 3

Lack of IL-6 signaling in canine OSA. The canine OSA cell line OSA16 was serum starved for two hours then stimulated with rcIL6 (30 ng/mL) for 0, 5, 10, or 30 minutes. Protein lysates were generated and separated by SDS-PAGE and Western blotting for pSTAT3 (Y705), total STAT3, pJAK2 (Y1007/1008), and total JAK2 was performed.

Figure 4

Src and STAT3 are associated with gp130 in human and canine OSA cell lines. Canine (OSA8) or human (SJSA) OSA cell lines were serum starved for two hours then left untreated or stimulated with rhOSM (50 ng/mL) for 15 minutes. Protein lysates were generated and immunoprecipitation performed for gp130. Co-precipitated proteins were separated by SDS-PAGE and Western blotting performed for total Src, total STAT3, gp130, or β-actin.

Figure 5

OSM does not alter the proliferation of OSA cell lines. Canine (OSA8) or human (SJSA) OSA cell lines were treated with 0, 50, or 100 ng/mL rhOSM for 72 hours in triplicate. Proliferation was analyzed using the CyQUANT cell proliferation assay kit. Proliferation values are listed as a percentage of PBS control and the bars represent the standard error of the mean.

Figure 6

OSM stimulation enhances MMP2 and VEGF activity and OSA cell invasion. A) Canine (OSA8) or human (SJSA) OSA cells were treated with 0, 50, or 100 ng/mL rhOSM or 100 ng/mL OSM in combination with the small molecule STAT3 inhibitor LLL3 (40 μM) for 72 hours. Media was collected, processed, and gel zymography performed as described previously [6]. B) Canine (OSA8) and human (SJSA) OSA cells were plated in serum free media with 50 ng/mL rhOSM in the upper wells of plates for invasion assays. Cells were incubated overnight to allow for invasion through a layer of Matrigel. The lower chamber of each treatment group contained either 10% fetal bovine serum alone (C10), C10 media with rhOSM (50 ng/mL), C10 media with rhHGF (50 ng/mL), or C10 media with both cytokines at 50 ng/mL. Cells were counted in ten random fields in quadruplicate replicates. The bars refer to the standard error of the mean. **p < 0.01; ***p < 0.001 C) Canine (OSA8) or human (SJSA) OSA cells were treated with 0, 50 ng/mL rhOSM, 50 ng/mL rhHGF, or the two cytokines together at 50 ng/mL for 72 hours. Media was collected, processed, and gel zymography performed as described previously. D) Canine (OSA8) and human (SJSA) OSA cells were left untreated or incubated with 50 or 100 ng/mL rhOSM, or 100 ng/mL rhOSM + LLL3 40 μM for 24 hours. Media was collected and VEGF concentrations determined by ELISA. E) Human (SJSA) OSA cells left untreated or incubated with 50 or 100 ng/mL rhOSM, or 100 ng/mL rhOSM + 40 μM LLL3 for 72 hours. Protein lysates were generated and separated by SDS-PAGE and Western blotting for VEGF and β-actin was performed.