Puringeric Signaling in Bone as a Potential Mechanism in Prostate Cancer Proliferation and Cancer‐Induced Bone Pain

Prostate cancer (PCa) is the second leading cause of cancer‐related deaths in men in the US. PCa bone metastasis occurs in approximately 2 out of 3 patients whose cancer has spread to other parts of the body. In particular the majority of PCa that metastasizes to bone results in osteosclerotic tumors. Osteosclerotic tumors arise from interactions between cancer cells and osteoblasts that results in increased bone formation, but reduced bone strength and increased mechanical strains. The increased load in bone resulting from tumor formation correlates with increased pain sensation. Cancer‐induced bone pain (CIBP) is one of the most detrimental symptoms to patients with bone‐localized cancer, greatly reducing patient's quality of life. Current treatment for CIBP is ineffective. A better understanding of the molecular mechanisms of CIBP is necessary for proper treatment. We propose that mechanical load of PCa cells results in ATP release which can activate local neurons through P2X 3 receptors resulting in CIBP. Additionally, we propose that nerve‐growth factor (NGF), a molecule implicated in PCa proliferation, enhances the potential for CIBP by through PCa cell proliferation and ATP signaling. To determine the effect of mechanical load on ATP release from PCa cells and activation of neurons, we mechanically loaded human bone‐derived PCa cells, C4‐2B4, via hypotonic swelling. Resultant C4‐2B4 load‐response conditioned media (L‐CM) +/− apyrase or various concentration of ATP were applied to primary mouse dorsal root ganglia (DRG) and changes in intracellular calcium were measured. To determine the role of P2X 3 in response to L‐CM, TNP‐ATP, a P2X 3 specific antagonist was utilized. Data indicated that mechanical load increased ATP release from C4‐2B4 cells and that L‐CM increased intracellular calcium in DRG which was reduced with apyrase. TNP‐ATP did not significantly block response to L‐CM suggesting another purinergic receptor may be responsible for this response. To address the role of NGF in PCa proliferation and purinergic signaling, we applied NGF to C4‐2B4 and counted cells daily for 4 days. To determine the effect of NGF on C4‐2B4 response to ATP, we applied various concentrations of ATP to C4‐2B4 cell pretreated with NGF for 48 hrs and measured intracellular calcium. NGF treatment demonstrated a 32% increase in cell number at day 4 when compared to control. Calcium imaging studies indicated that response to ATP varied with addition of NGF indicating a potential pathway for altered sensitivity to ATP. These studies indicate that NGF may play an important role in the growth of metastatic prostate cancer cells in bone. Taken together these data indicate purinergic receptors as a potential therapeutic target for CIBP. Support or Funding Information Delaware INBRE program NIH‐NIGMS (8 P20 GM103446‐15) Charles Peter White Fellowship