Prediction Of Bone Loss And The Application Of ARRY-614 In The Treatment Of Breast Cancer Osteolysis By Inhibiting Bone Loss

BackgroundOsteoclast,as the main functional cell of bone absorption,plays an important role in the process of bone development,growth,repair and reconstruction.Osteoclasts are formed by the fusion of bone marrow monocytes/macrophages(BMMs),whereas osteoblasts are derived from mesenchymal stem cells.The differentiation of progenitors into osteoclasts requires critical cytokines,including receptor activator of nuclear factor-K B ligand(RANKL)and macrophage colony-stimulating factor(M-CSF).RANKL is the key cytokine for osteoclast differentiation,while M-CSF is necessary for the survival and proliferation of osteoclast precursors.Binding of RANKL to its receptor RANK leads to the trimerization of RANK and the recruitment of adaptor molecules,such as tumour necrosis factor receptor-associated factor 6(TRAF6),followed by the activation of signalling pathways,including the mitogen-activated protein kinase(MAPK)and nuclear factor kappaB(NF-κB)pathways.Downstream regulators such as c-Fos and nuclear factor of activated T cells c1(NFATc1)are subsequently upregulated and activated,resulting in the formation of osteoclasts.Under physiological conditions,there is a balance between osteoblast-mediated bone resorption and osteoblast-mediated bone formation to maintain normal bone mass.If this balance is disturbed,conditions such as focal osteolysis caused by bone metastases,osteoporosis,or osteosclerosis can occur.Breast cancer is the most common cancer in women,and the skeleton is the most common target tissue for breast cancer metastases.Breast cancer cells can directly secrete the receptor activator of nuclear factor-κB ligand(RANKL),or cytokines such as the parathyroid hormone-related protein,tumor necrosis factor(TNF)alpha,macrophage colony-stimulating factor(M-CSF),and interleukin-1,which stimulate RANKL expression on the surface of osteoblasts and in the bone matrix.Excessive activation of osteoclasts is considered to be the main cause of osteolysis.Currently,bisphosphonates remain the mainstay in treating breast-induced bone diseases.Unfortunately,many studies have shown that the long-term use of bisphosphonates can inhibit the natural regeneration of bone tissue,causing non-spinal fractures in patients such as delayed union and femoral shaft fractures.Thus,it is important to develop alternative therapeutic agents for treating breast cancer-induced osteolysis.The MAPKs are a class of serine/threonine kinases involved in many cellular activities such as cell proliferation,differentiation,invasion,migration,and death.p38 MAPK is responsive to RANKL and is involved in osteoclast differentiation.Moreover,the p38 MAPK-mediated signaling pathway plays an indispensable role in breast cancer bone metastasis.ARRY-614 is a novel inhibitor of p38 MAPK and has been investigated in phase I clinical trials for treating myelodysplastic syndrome.However,the effect of ARRY-614 on breast cancer induced osteolysis is not be explored.Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density(BMD)and deteriorated microarchitecture.The microstructure and strength of trabecular bone deteriorated as the decreasing of bone mineral density.The mechanism of osteoporosis and osteopenia is still controversial.Lots of researches showed that the peripheral blood mononuclear cells may be closely related to osteoclasts and include osteoclast precursors.Peripheral blood mononuclear cell is the important resource of osteoclasts.Meanwhile,osteoclast and osteoclast precursor played an important role in bone metabolism.So,the peripheral blood mononuclear cells count maybe correlated to bone health.However,as far as we know,there is no research concentrated on the relationship between the peripheral blood mononuclear cells count and bone health.The exact relationship between the peripheral blood mononuclear cells count and bone health is still controversial.Objective1.We aimed to elucidate the role and underlying mechanism by which ARRY-614 on breast cancer induced osteolysis treatment.2.To explore whether there is a correlation between the peripheral blood mononuclear cell count and bone health through multi-center study.To clear and define the explicit relationship between the peripheral blood mononuclear cell count and bone health.To clarify whether some parameters would influence peripheral blood mononuclear cell count and bone health.Evaluating whether peripheral blood mononuclear cell count could be used to screening osteoporosis and guide further dual Dual-energy x-ray absorptiometry examinationMethods:1.The effect of ARRY-614 on osteoclast formation and bone resorption were assessed by staining for tartrate-resistant acid phosphatase(TRAP)and measuring resorption of bovine bone slices in vitro.The expression levels of nuclear factor of activated T cells 1(NFATc1),dendritic cell-specific transmembrane protein,TRAP,DC-STAMP,CTSK were measured via the real-time polymerase chain reaction.The effects of ARRY-614 on breast cancer cell proliferation,apoptosis,migration and invasion were explored by the cell counting kit 8,flow cytometry,and transwell assays respectively.Western blotting was conducted to detect the effects of ARRY-614 on p38-mediated cAMP-response element-binding(CREB)and signal transducer and activator of transcription(STAT)3 activation during osteoclastogenesis.Mouse models were established to explore the effects of ARRY-614 on both titanium particle-induced osteolysis and breast cancer-associated osteolysis in vivo.2.This research included 2806 community men aged 50 years who underwent full health examinations from October 2007 through December 2011 in four medical centers located in Zhejiang province southeast of China.Baseline characteristics were all recorded using a standardized questionnaire.Continuous variables were compared using the Wilcoxon signed-rank test or Mann-Whitney U test as appropriate;categorical variables were compared using the x2 test or Fisher’s exact test.The statistical results were presented as the mean standard deviation.Analysis of covariance and multiple linear regression were used to examine the relationship between the peripheral blood mononuclear cell count,bone mineral density value,and T-score.Multivariate analysis was performed to further confirm the association between the peripheral blood mononuclear cell count and bone health after considering the above confounding factors(Model 1 included peripheral blood mononuclear cell count,neutrophil count,eosinophil count,basophil count,lymphocyte count,red blood cell count,platelet count,age,height,weight,SBP,and glucose;Model 2 included peripheral blood mononuclear cell count,neutrophil count,eosinophil count,basophil count,lymphocyte count,red blood cell count,platelet count,age,height,weight,SBP,glucose,serum calcium,serum phosphorus,and alkaline phosphatase;Model 3 included peripheral blood mononuclear cell count,neutrophil count,eosinophil count,basophil count,lymphocyte count,red blood cell count,platelet count,age,height,weight,SBP,glucose,serum calcium,serum phosphorus,alkaline phosphatase,aspartate transaminase,and alanine aminotransferase;Model 4 included peripheral blood mononuclear cell count,neutrophil count,eosinophil count,basophil count,lymphocyte count,red blood cell count,platelet count,age,height,weight,SBP,glucose,serum calcium,serum phosphorus,alkaline phosphatase,aspartate transaminase,alanine aminotransferase,estimated glomerular filtration rate,and total protein.).Because BMD is also affected by obesity and hypertension,we created further subgroups based on BMI(BMI<25 and BMI>25)and SBP(SBP<140 and SBP≥140).A two-sided P-value<0.05 was considered statistically significant.Results1.In our study,we demonstrated that a novel p38 inhibitor ARRY-614 inhibited RANKL-induced osteoclast formation and bone resorption in vitro.P38-mediated STAT3 activation was impaired by ARRY-614,leading to decreased NFATc1 expression during osteoclastogenesis.Furthermore,ARRY-614 exerted anti-tumor effects in breast cancer cells in vitro and MMP-2/-9 expression was impaired by ARRY-614 via suppressing p38-mediated CREB and STAT3 activation.Importantly,ARRY-614 suppressed titanium particle-induced osteolysis and breast cancer-associated osteolysis in vivo.2.A total of 2806 subjects were enrolled.According to exclusion criteria,481 elderly men were eliminated;2325 participants were included in the analysis,569 in the at least osteopenia group and the remainder in the control group.The peripheral blood mononuclear cell count was significantly high among subjects with "at least osteopenia"compared with controls.In analysis of covariance adjusted for potential confounders,the bone mineral density value and T-score had a significant decreasing trend across the quartiles of peripheral blood mononuclear cell count.In univariate analysis,the peripheral blood mononuclear cell count had a strong association with "atleast osteopenia"(odds ratio[OR]=2.520,95%confidence interval[CI]:1.397-4.547).Conclusion1.ARRY-614 may be a promising drug for repositioning toward the treatment of breast cancer-induced osteolysis.X2.The peripheral blood mononuclear cell count is significantly associated with bone loss in elderly men and the exact mechanism requires further clarification.