Mechanism Investigation Of GPR84 On The Osteoclasts In Bone Metastasis Of Colorectal Cancer

Research background and purpose:Colorectal cancer is currently the fourth most common cancer in the world,accounting for ten percent of all annual diagnosed and cancer-related deaths worldwide,with approximately 900,000 deaths annually.Liver and lung are the most common metastatic sites in patients with advanced CRC,and bone is the third metastatic site,with a lower incidence than that of others.However,skeletal-related events(SREs)such as pathological fractures,pain,and hypercalcemia caused by bone metastasis are the main causes of cancer-related deaths.Bone metastasis produce negative affects on quality of life and decrease overall survival.With the advent of China’s aging society,the incidence of colorectal cancer has gradually increased,and the medical burden has also increased accordingly.As the advancement of surgical technology and medical facilities,the continuous emergence of antineoplastic drugs and the application of high-precision radiotherapy,the survival time of patients with colorectal cancer continues to improve.Treatment for the bone metastasis of colorectal cancer has gradually become the research hotspot.As with most primary and metastatic malignancies,bone destruction is closely associated with the activation of osteoclasts and osteoclast/osteoblast imbalance.Osteoclasts originate from bone-derived mononuclear macrophages and are abnormally activated in the tumor microenvironment,which result in the imbalance of osteoblasts-osteoclasts and ultimately mediating bone destruction.Breast,lung and prostate cancer are the three most common malignancies of bone metastasis.There have been a lot of studies on how osteoclasts participate in bone destruction.However,discrepancies exist in occurrence,development and metastasis of different malignant tumors,which means that the cell components,cytokine types and contents in the tumor microenvironment may also be different.However,there is few studies on how osteoclasts are involved in bone metastasis of colorectal cancer.G protein coupled receptor(GPRs)is a large class of membrane proteins,which plays an important role in mediating the cell response to external stimuli,and are involved in numerous physiological and pathological activities,including growth,hormonal homeostasis,regulation of metabolism,feeding behavior,and mediation of inflammatory responses.GPRs particiapte in the interaction between cancer cells and normal cells,and some members of the GPR family have been reported to be involved in tumor metastasis.In bone metastasis,ovarian cancer GPR1 mediates communication between cancer cells and osteoblast-like cells.The expression of GPR65/ADGRG1 was upregulated in 4T1 cells of breast cancer cell line and in bone metastases.GPR65 is downregulated in OCPs during bone metastasis of CRC by mi R-7062-5p.GPR120 increases the motility and aggressive activity of pancreatic cancer cells,while GPR40 works just the opposite.GPR84 is a member of GPRs family which is described as a pro-inflammatory receptor and highly expressed in monocytes/macrophages.Monocytes/macrophages are the source of osteoclasts,and GPR84 overexpression has been shown to negatively regulate RANKL-induced osteoclast differentiation through NF-κB and MAPK signaling pathways.However,the expression of GPR84 in bone metastases of colorectal cancer,whether it is involved in tumor progression,and its effect on osteoclasts and mechanism are remain unclear.Here,we investigate the role of GPR84 in bone metastasis of colorectal cancer,providing idea for finding potential therapeutic targets.Materials and Methods:1.The tibial metastasis model of mouse colon cancer was constructed by using MC-38 cells.The mice were killed on day of 0/3/7/14 to obtain BMMs(bone-derived monocytes and macrophages).q RT-PCR analysis was performed to detect the relative expression of GPR84 m RNA at different time points.Immunofluorescence assay was used to determine the relative fluorescence intensity of GPR84 at each time points.The expression of GPR84 protein at each time point was verified by Western-Blot assay.2.Normal mouse bone marrow cells were collected and cultured in medium,containing M-CSF.The treatment group were added with GPR84 agonist6-OAU,while control group treated with PBS,and then antigen-antibody incubation,Brdu were labeled for flow cytometry analyzing.q RT-PCR was applied to detect the m RNA expression of cell cycle biomarkers Cenpa,Cdc20,Cdk1,Ccnd1.3.The BMMs were extracted from of normal mice and cultured in CM(condition medium)which derived from MC-38 cells,and RANKL/M-CSF were added for osteoclast induction.BMMs were treated with plasmid 6-OAU and overexpressed GPR84,TRAP staining and the number of osteoclasts was detected by,and then TRAP staining was utilized for determining the number of osteoclasts.Two classic osteoclast biomarkers were detected by q RT-PCR assay.4.BMMs were obtained and cultured in α-MEM M-CSF(50 ngm L)RANKL(100ngm L)medium,containing 10% fetal bovine serum and 1% penicillin-streptomycin solution,and then transfected with plasmid overexpressing with GPR84.The level of P38/JNK/ERK phosphorylation were detected by Western-blot assay.The JNK ERK pathway were activated by using anisomycin t BHQ,and the number of osteoclasts were verifyed by TRAP staining.5.BMMs from normal mice were cultured in CM medium for 48 h,and the expression GPR84 m RNA was determined by q RT-PCR.BMMs were cultured in growth medium and treated with EGF,IL-11,CTGF,PTHr P respectively.q RT-PCR was used to determine the m RNA expression level of GPR84.BMMs cultured in CM and treated with IL-11 Nab to treat IL-11,and then detected the expression of GPR84 m RNA by qrt-pcr assay.In the animal experiment,IL-11,IL-11+6-OAU,IL-11 Nab were injected into the tibial bone marrow of mice with bone metastasis of colon cancer,and then the expression of GPR84 m RNA was determined by qrt-pct.TRAP staining was performed in IL-11 group and IL-11+6OAU group for verifying the number of osteoclast.6.BMMs was cultured in CM medium and treated with IL-11/IL-11 Nab.The phosphorylation level of STAT1 and the protein level of GPR84 were detected by Western-Blot.The BMMs transfected with plasmid overexpressing STAT1 and cultured in CM medium,and treated with IL-11,the q RT-PCR was applied to detect the m RNA expression GPR84.7.In the colon cancer mouse model,the mouse were treated with GPR84 agonist in experiment group,while PBS was applied in control groups.Three weeks later,the tibias of mice was obtained for TRAP staining to determine the effect of 6-OAU on the expression of osteoclasts in animal experiments.μ-CT was used to detect the related parameters of tibia,including: BMD,BV/TV,Tb.Th,Tb.N,Tb.Sp.Result:1.GPR84 was expressed in normal mouse BMMs,and it’s m RNA and protein expression levels were downregulated as the advancement of CRC bone metastasis.2.GPR84 had no effect on the proliferation of mononuclear macrophages(BMMs),but the activation or overexpressing of GPR84 could decrease the number of osteoclast and downregulate the m RNA expression of osteoclastogenesis biomarker significantly.3.The overexpression of GPR84 decreased the phosphorylation level of JNK,ERK and p38 MAPK pathways,but the activation of JNK and ERK pathways could increase the number of osteoclasts.4.Of the four common cytokines secreted by tumor cells,only IL-11 inhibits the m RNA expression of GPR84,but the application of IL-Nab could reverse this effect.Animal experiments have shown that tumor cells-derived IL-11 partially inhibits the expression of GPR84 in early OCPS and increases the number of osteoclasts,and activating of GPR84 can reverse this effect.5.Tumor cell-derived IL-11 inhibits the phosphorylation of STAT1 and the expression of GPR84.6.Animal experiments preliminarily confirmed that Activation of GPR84 inhibited osteoclastogenesis and alleviated bone destruction caused by tumor bone metastasis.Conclusion:The preliminarily research have shown that GPR84 negatively regulated osteoclast ogenesis through MAPK pathway,and tumor cell-derived IL-11 partially inhibitd GPR84 expression by inhibiting the activation of STAT1.The activation of GPR84 prevented osteoclast generation in the tumor microenvironment and alleviated bone destruction caused by CRC bone metastasis.