The Role Of Spinal CREB/CRTC1 Signaling In The Maintenance Of Bone Cancer Pain

Background and Objective:Pain is one of the most common symptoms presented by patients with cancer.The epidemiological survey indicated that approximately 90%of cancer patients suffer pain during the course of disease.Notwithstanding Non-opioid analgesics,opioids and adjuvants administered following the World Health Organization's three-step "therapeutic ladder" for cancer pain relief,pain associated with cancer is treated inadequately or difficult to manage in about 45%of all cancer patients.Bone cancer pain is a severe and relatively common type of cancer pain,and the relief of bone cancer pain is often unobtainable and continues to be a challenging endeavor.Patients with primary bone sarcomas or malignant tumors that have metastasized to bone are frequently confronted with poor quality of life.Therefore,it is essential to investigate the basic mechanisms that generate and maintain bone cancer pain and find novel and effective analgesics to block this debilitating pain.Previous studies demonstrated that N-methyl-d-aspartate receptor(NMDAR)in spinal dorsal horn plays an essential role in various pain models including bone cancer pain.Nociception-induced Ca2+ influx via NMDARs increases the phosphorylation of cyclic adenosine monophosphate(cAMP)response element binding protein(CREB)at Serine-133 site(p-CREB)to initiate the downstream target genes transcription is considered to be an important response that mediates the initiation and maintenance of central sensitization.Recent investigations indicated that CREB-regulated transcription coactivator 1(CRTC1)is necessary for CREB-mediated genes transcription and dramatically increases CREB-mediated transcriptional activity.CREB/CRTC1 signaling-dependent target genes including Brain-derived neurotrophic factor(BDNF),NMDA receptor subunit 2B(NR2B)and microRNA-212/132 mediate activity-dependent synaptic plasticity and in turn loop back to amplify CREB/CRTC1 signaling,which may form a positive feedback signaling circuit.However,the role of CREB/CRTC1 signaling in the maintenance of bone cancer pain is not quite understood.This study aimed to investigate the role of CREB/CRTC1 signaling in bone cancer pain using a RNA interference method to provide further insight into the mechanisms and treatment of bone cancer pain.Methods:The right femur marrow cavity of C3H/HeNCrlVr male mice were inoculated with NCTC 2472 fibrosarcoma cells to establish bone cancer pain model and the equal volum of a-minimal essential medium(?-MEM)without NCTC 2472 fibrosarcoma cells were injected into the intramedullary space of the right femurs in the sham mice.The number of spontaneous flinches/2 min(NSF)and paw withdrawal mechanical threshold(PWMT)were used to assess pain behaviours performance.The effects of repeated intrathecal administration with Adenoviruses expressing CREB-siRNA or CRTC1-siRNA on nociceptive behaviors were further investigated.Western blotting and real time PCR were applied to examine the expression of CREB,CRTC1 and CREB/CRTC1-target genes in the L3-L5 spinal cord segments.Immunofluorescence was applied to detect the expression of glial fibrillary acidic protein(GFAP)in the L3-L5 spinal dorsal horn to reflect astrocytes activation level.Results:(1)Inoculation of NCTC 2472 fibrosarcoma cells into the right femur of the male C3H/HeNCrlVr mice induced progressive spontaneous flinches and mechanical hyperalgesia(P<0.05);(2)The expression of p-CREB and CRTC1 in the L3-L5 spinal cord was progressively up-regulated during the development and maintenance of bone cancer pain(P<0.05);(3)Repeated intrathecal administration with Adenoviruses expressing CREB-siRNA or CRTC1-siRNA improved bone cancer pain behaviours,and inhibited the up-regulation of CREB/CRTC1-target genes in spinal cord,including NR2B,BDNF and miR-212/132(P<0.05);(4)Results of Immunofluorescence staining indicated that inoculation of NCTC 2472 fibrosarcoma cells into the right femur induced the activation of astrocytes in the L3-L5 spinal dorsal horn,characterized by the up-regulation of GFAP,and inhibition of spinal CREB/CRTC1 signaling decreased the expression of GFAP in spinal dorsal horn(P<0.05).Conclusions:Activation of CREB/CRTC1 signaling in spinal cord may play an important role in the maintenance of bone cancer pain.Inhibition of spinal CREB/CRTC1 signaling and interruption to the positive feedback circuit between CREB/CRTC1 and its targets,inhibited the spinal astrocytes activation,and reduced bone cancer pain effectively.