The contribution of stem cell factor and its receptor c-Kit to cancer-induced bone pain
Kelly F. Contino, Jenna Ollodart, Yang Yu, Sun H. Park, Shunsuke Tsuzuki, Kara Rollins, Tyler M. Heethouse, Joshua Chu, Laiton R. Steele, Takahiro Kimura, Jingyun Lee, Cristina M. Furdui, Lance D. Miller, Fang-Chi Hsu, Yusuke Shiozawa
Kelly F. Contino, Jenna Ollodart, Yang Yu, Sun H. Park, Shunsuke Tsuzuki, Kara Rollins, Tyler M. Heethouse, Joshua Chu, Laiton R. Steele, Takahiro Kimura, Jingyun Lee, Cristina M. Furdui, Lance D. Miller, Fang-Chi Hsu, Yusuke Shiozawa
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Research Article Neuroscience Oncology

The contribution of stem cell factor and its receptor c-Kit to cancer-induced bone pain

Abstract

Cancer-induced bone pain (CIBP) is among the most common and debilitating symptoms in patients with bone metastasis. Current treatments are somewhat effective but have severe side effects. For the future development of safer CIBP treatments, in this study, we sought to investigate the mechanisms whereby the nerve-cancer interaction controls CIBP. We found that c-Kit, a receptor tyrosine kinase, was activated in the dorsal root ganglia (DRG) sensory neurons of mice with CIBP and that c-Kit’s sole ligand, stem cell factor (SCF), was enhanced in the bone marrow with bone metastasis. When DRGs were treated with SCF or conditioned medium from high SCF-expressing cancer cells, in vitro nerve sprouting was enhanced, and this effect was abolished with c-Kit inhibitors. Mice inoculated intrafemorally with cancer cells that had varying levels of SCF expression developed CIBP and enhanced peripheral nerve sprouting in an SCF-dependent manner. Downstream proteomic analysis revealed that SCF upregulated and activated fibroblast growth factor 1 (FGF1) in DRGs. When FGF1 was knocked down in DRGs, SCF-mediated nerve sprouting was prevented. Taken together, our studies demonstrate the importance of the SCF/c-Kit axis in CIBP and nerve sprouting and identify the SCF/c-Kit/FGF1 pathway as a potential therapeutic target for CIBP.

Authors

Kelly F. Contino, Jenna Ollodart, Yang Yu, Sun H. Park, Shunsuke Tsuzuki, Kara Rollins, Tyler M. Heethouse, Joshua Chu, Laiton R. Steele, Takahiro Kimura, Jingyun Lee, Cristina M. Furdui, Lance D. Miller, Fang-Chi Hsu, Yusuke Shiozawa

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Figure 5

The SCF/c-Kit axis is responsible for in vitro nerve sprouting.

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The SCF/c-Kit axis is responsible for in vitro nerve sprouting. (A) Repr...
(A) Representative IF images of β3-tubulin–positive (a pan neuronal marker) neurite outgrowth of murine primary DRG cells treated with SCF. Original magnification, ×10. Scale bar: 100 μm. (B) Quantification of neurite outgrowth in A. Mean ± SEM. Student’s t test (**P ≤ 0.01). (C) Representative single-cell traces showing calcium changes in murine primary DRG cells incubated with Fura-2 in response to treatment with vehicle or SCF (24 hours) prior to depolarization with high KCl (50 mM). Fluorescent signals are scaled as ΔF/F0 and Fura-2 fluorescent signals are presented as 408/510 nm. (D) Quantification of peak calcium influx by change in fluorescent intensity shown in C. Mean ± SEM. Student’s t test (not significant). (E) Neurite outgrowth of murine primary DRG cells treated with serum-free growth medium (control conditioned medium [CM]) or CM obtained from B16-F10, RM-1, or LL/2 cells. Mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons (**P ≤ 0.01, ***P ≤ 0.001). (F) Neurite outgrowth of murine primary DRG cells treated with CM obtained from SCF-overexpressing B16-F10 (B16-F10 SCF OE) and SCF-downregulated RM-1 (RM-1 SCF knockdown [KD] or SCF-downregulated LL/2 [LL/2 SCF KD]) vs. CM obtained from respective control cells that were transfected with empty vector (EV). Mean ± SEM. Student’s t test (*P ≤ 0.05). (G and H) Neurite outgrowth of murine primary DRG cells treated with or without LL/2 cell CM in the presence or absence of c-Kit inhibitors, (G) ISCK03 and (H) repretinib.