Lung Adenocarcinoma Cells Promotes Osteoclast Differentiation Via Jagged1/Notch Signaling

Background and Objectives:Cancer metastasis is responsible for the major mortality of patients suffered fromcancer. Many malignant tumors, such as the lung cancer, prostate cancer and breast cancer,have the tendency of metastasis to bone. It has been reported that approximately30-40%patients with lung cancer posses bone metastasis at the end stage of the diseases.Patienswith lung cancer bone metastasis are most frequently characterized of seriously osteolyticbone destruction. Meanwhile, they are often subjected to the skeletal related eventsincluding severe bone pain, pathological fractures, hypercalcemia, and spinal cordcompression syndrome, etc. So it is very significative to prevent and cure the cancermetastasis to bone. The cancer cells invade into the microenviroment of bone marrow andsecrete large amounts of cytokines, then exert influence on host cells(such ashematopoietic stem cell, osteoclasts, osteoblasts) and bone marrow stromal each other,creating a so-called vicious cycle. Disruption of this interaction will allow us to designspecific therapeutic interventions for bone metastases, but the particular molecularmechanism is poorly understood so far.Jagged1is one of the ligand of Notch, binding with its specifical receptor activatesNotch signaling pathway and then play biologic role. Notch signaling pathway could beblocked by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT).Notch signaling play an important role in the embryonic development, which can influencemany cellular processes, including adult cell differentiation, proliferation, ect. Abnormalactivation of Notch also correlates to the occurrence and development of many disease. Inrecent years, Jagged1/Notch signaling has been found to play important roles in the cancerdevelopment, progression and metastasis. Indeed, the prostate or breast cancer patients whohave the high level expression of Jagged1often exhibit poor biological behaviour prognosis,and so are the non-small cell lung cancer patients. It has been reported that Notch signaling could mediate the differentiation and maturation of osteoclast precursors underphysiological state, but effects of Jagged1/Notch signaling on osteoclast precursors underpathological conditions, such as bone metastasis of lung cancer, have not been reported.Therefore, the expressions of Jagged1in human lung cancer patients were detected inthe present study, then the role of Jagged1on the osteoclast differentiation and proliferationof CD14positive monocyte was investigated. At last, we assessed the hypothesis thatwhether lung cancer cells line A549conditioned medium(CM) could mediate osteoclastdifferentiation and proliferation through Notch signaling or not. The research wouldfacilitate our better understanding the underlying mechanism of lung cacer osteolytic bonemetastasis, and may provide a new therapeutic targets to patients suffered lung cancermetastasis to bone.METHODS:Firstly, the expression of Jagged1in metastases and tissue adjacent to carcinoma oflung cancer patients was detected. Then the effects of Jagged1on osteoclast differentiationand proliferation of CD14positive monocyte were assessed. At last, lung cancer cell lineA549CM was collected, and the effect of Jagged1/Notch in A549CM induced osteoblasticdifferentiation and proliferation by co-culture system was evaluated with or without Notchsignaling inhibitor.Effects of Jagged1on osteoclast differentiation and proliferation of CD14positivemonocyteThe expression of Jagged1in metastases and tissue adjacent to carcinoma of lungcancer patients was detected by immunohistochemical method. CD14positive monocyteswere obtained from human peripheral blood by Ficoll density gradient centrifugationcombined with magnetic beads separation, and were incubated with α-MEM containingmacrophage colony-stimulating factor (M-CSF) as the Control group. Jagged1recombinantprotein was supplied in Jagged1group. The Notch pathway blocking agent DAPT was usedto treat monocytes with Jagged1in DAPT group. The expression levels of osteoclast markergenes tartrate-resistant acid phosphatase (TRAP), cathepsin K (CK), calcitonin receptor(CTR) and Notch key target genes hairy and enhancer of split-1(Hes-1) and HES-relatedwith YRPW motif-1(Hey-1) were measured by real-time fluorescence quantitative PCR.The TRAP staining was used to detect the formation of osteoclast, and the bone resorption was evaluated by scanning electron microscope. The proliferation of CD14positivemonocytes was detected by cell counting kit-8(CCK-8) method.Role of Jagged1/Notch signaling in the regulation of A549CM on osteoclastdifferentiation and proliferation of CD14positive monocyteExperiment was divided into three groups: Control group, A549CM group and DAPTgroup. CD14positive monocytes were incubated with α-MEM containing macrophagecolony-stimulating factor (M-CSF) as the Control group. The A549CM(10%V/V) wasadded to incubated CD14positive monocytes with M-CSF in A549CM group. DAPT wasused to treat monocytes with A549CM, M-CSF in DAPT group. The mRNA expressionlevels of TRAP, CK, CTR, Hes-1and Hey-1were measured by real-time fluorescencequantitative PCR. The formation of osteoclast was detected by TRAP staining, and the boneresorption was evaluated by scanning electron microscope. The proliferation of CD14positive monocytes was detected by CCK-8method. The fluorescent expression of activefragment NICDwas determined by immunofluorescence.RESULTS:Jagged1promoted osteoclast differentiation and inhibited proliferation byactivating Notch signaling pathway1. High expression of Jagged1in bone metastases specimens was observed in contrastof no expression or low expression in adjacent tissues.2. TRAP, CK and CTR mRNA expression levels of CD14positive monocytes weresignificantly higher in Jadded1group than those of Control group and DAPT group.3. The number of TRAP positive multinuclear cell was significantly higher in Jagged1group when compared with Control and DAPT group.4. The area of bone resorption of CD14positive monocytes was strengthenedobviously by Jagged1.5. Cell proliferation of CD14positive monocytes was inhibited in Jagged1group.6. The expressions of Notch key target genes Hes-1and Hey-1mRNA weresignificantly increased in Jagged1group than those of Control group and DAPT group.A549CM promoted osteoclast differentiation by activating Notch signaling.Meanwhile the proliferation of CD14positive monocytes was promoted by A549CM,which would be further heightened by DAPT. 1. The mRNA expression levels of TRAP, CK and CTR in A549CM group wereupregulated as compared with Control group.2. A549CM significantly increased the number of TRAP+multinuclear cells via Notchsignaling in comparison with Control group and DAPT group.3. The area of bone resorption was obviously higher in A549CM group, which wasrelated to Notch signaling.4. The up-regulation of CD14positive monocytes proliferation induced by A549CMwas further promoted by DAPT.5. The mRNA expression levels of Hes-1and Hey-1in A549CM group were higherthan those in DAPT group and Control group.6. The nuclear fluorescence intensity of NICD in A549CM group were upregulated ascompared with Control group and DAPT group.CONCLUSIONS:1. There is a positive correlation between the expression level of Jagged1and lungcaner bone osteolytic injury. The expression of Jagged1in bone metastases is higher thanthose of adjacent tissues.2. Jagged1promotes osteoclast differentiation and inhibits proliferation by activatingNotch signaling pathway.3. The CM of lung cancer A549cells can promote osteoclast differentiation byactivating Notch signaling. Notch signaling would provide a novel therapeutic target forlung cancer-induced osteolytic lesion.