Effects Of CXCR4~+ Non-small Cell Lung Carcinoma Cells On Osteoclastogenesis And The Underlying Mechanism

BackgroundWith the aging of the population,the number of lung cancer patients and people died of cancer increased vastly.In China,the leading cause among cancers are lung cancer,which includes small cell lung carcinoma（SCLC）and non-small cell lung carcinoma（NSCLC）.SCLC accounts for 15%while the rest are most of NSCLC.Lung cancers are most likely to spread to the bone,the percentage is up to 30-40%for late-stage lung cancer patients.The bone metastases are often associated with complications,so-called skeletal-related events（SREs）,defined as pathologic fracture,spinal cord compression,the pain of the bone and hypercalcemia of malignancy.The development of SREs inversely correlates with quality of life and survival time in affected patients.The bone metastases could be briefly considered as two stages,transcoelomic metastasis and colony growth.Tumor cells,osteoblasts,osteoclasts,bone extracellular matrix,the bone microenvironment and the niche are main components of a vicious cycle necessary for the initiation and development of metastatic lesions in the skeleton.Previous research has shown CXCL12-CXCR4 axis plays an important role in the development of tumor metastasis.In breast cancer,the tumor stromal cells select CXCR4⁺cells through secreting CXCL12 which lead to target organ metastases.The percentage of CXCR4⁺NSCLC cells is higher in skeleton tumor.Blockade of CXCL12-CXCR4 axis could inhibit the metastases of NSCLC including bone metastasis.In the lung tumor A549 cell lines,knockdown of CXCR4 with RNAi decreased the tumor metastases ability.Therefore,CXCL12-CXCR4 participates the porcess of NSCLC bone metastases.Metastasis to the bone needs to go through metastatic quiescence,formation of tumor cell niches and finally tumor cell growth to sites.The metastatic tumor cell often successfully break the balance between osteolytic and osteogenic programs.Following osteoclast maturation,the“vicious cycle”of lytic bone metastasis takes place whereby degradation of the bone matrix by osteoclasts releases various growth factors such as TGFβ,FGFs and EGFs,promoting tumor cell growth.However,it remains unknown that how the lung cancer cells break the osteolytic/osteogenic axis,induce the osteoclastogenesis and start the bone lysis to facilitate the formation of bone metastases.VCAM-1 is considered to contribute to the initiation of bone lysis during breast cancer metastasis.Intriguingly,whether VCMA1 is involved in the development of lung cancer bone metastasis needs further investigation.VCAM-1（vascular cell adhesion molecule 1,CD106）belongs to a transmembrane immunoglobulin family and is mainly expressed in stimulated endothelial cells.The VCAM-1 could be catalyzed to soluble VCAM-1.The main receptor of VCAM-1 isα4β1（very late antigen-4,VLA-4）,which was expressed in many hematopoietic cells including lymphocytes,monocytes and granulocyte.According to studies in breast cancer,VCAM-1is up-regulated and involved in the micro-environmental interaction between tumor cells and stromal cells.VCAM-1 facilitates the metastasis of breast cancer cells to lung and bone.According to previous study,tumor cells adhere to pulmonary vessels,activate endothelial VCAM-1 expression,recruit bone marrow cell and promote the survival of tumor cell.Moreover,VCAM-1 is upregulated in certain NSCLC tumor tissue.Compared with healthy control population,the serum VMCA-1 levels in NSCLC patients are significantly higher.VCAM-1 correlates a higher chemotherapy sensitivity and survival rates.In myeloid tumor study,the tumor cells interact with the stromal cells via the ligand-receptor interaction between VCAM-1 andα4β1.However,the role of VCAM-1 in the bone metastasis of lung cancer has not been reported.Based on the above,we speculate that VACM-1 might participate the metastasis of lung cancer,especially in the bone metastasis and the lysis of bone.Here,in this study we aimed to establish the CXCR4⁺NSCLC cell lines and mimicked the CXC12-rich tumor-bone microenvironment.Further,we explored the mechanism of how CXCR4⁺NSCLC cells activate the osteoclast and initiate the lysis of bone.Hopefully,we expected our research could provide a potential therapeutic target for NSCLC metastasis to bone.Methods1.Determination of CXCR4 expression in the bone metastasis tissue of NSCLC and different NSCLC cell lines1.1 Immunohistochemistry staining detected CXCR4 in the bone metastasis tissue of lung adenocarcinoma,and TRAP staining detected OCs in tumor-bone inerface.1.2 CXCR4,CXCR7 and CXCL12 expression was measured with real time PCR and Western Blot in A549,NCI-h1299,and H1975 cell lines.2.Construction and identification of CXCR4⁺or CXCR4^-NSCLC cellLentivirus vector containing shRNA against CXCR4 were constructed and transfected into NCI-H1299 cell lines.Lentivirus containing CXCR4 genes were also constructed and transfected into H1975 cell lines to establish CXCR4⁺NSCLC cell lines.2.1 The expression of CXCR4 were determined with real time PCR and Western Blot in A549,NCI-H1299,H1975 cell lines transfected with corresponding lentivirus.2.2 The cell proliferation of NSCLC with different expression level of CXCR4 were detected with CCK-8 Kit.2.3 Wound scratch assay and transwell migration chamber was exploited to measure the effects of CXCR4 expression on NSCLC migration/invasion.3.Effects of CXCR4⁺NSCLC cell on osteoclastogenesisThe RAW 264.7 monocyte cell line is the most commonly used osteoclast precursor.Low dose RANKL and NSCLC conditional medium were used to induce RAW 264.7 into osteoclast.NSCLC cells were used to explore the effects of CXCL12-CXCR4 axis on osteoclast differentiation.3.1 The mRNA levels of osteoclast marker genes such as TRAP,CK were measured with RT-PCR.3.2 Staining of TRAP were used to identify osteoclast differentiation and maturation.3.3 AMD3100,a CXCR4 antagonist was applied to detect the effects of CXCR4 on osteoclast differentiation and maturation.4.Mechanism of CXCR4⁺NSCLC cell promoted osteoclastogenesis4.1 ELISA kits were used to measure the cytokine during conditional medium induced osteoclast differentiation.4.2 Real time PCR and Western Blot were exploited to determine the gene expression in H1975 cell lines.4.3 The mRNA levels of osteoclast marker genes such as TRAP,CK were measured with RT-PCR.4.4 Staining of TRAP was used to measure osteoclast differentiation and maturation.4.5 ADAM17 antagonist including TAPI-1 and TAPI-2 were added to the conditional medium to determine its effects on osteoclast differentiation and maturation.Results1.CXCR4 expression in the bone metastasis tissue of NSCLC and different NSCLC cell lines1.1 CXCR4 is stained positive in the bone metastasis tissue of NSCLC,especially in the area the bone was severely invaded,and the number of OCs is correlation with CXCR4.1.2 CXCR4 is highly expressed in NCI-H1299 while the expression in H1975 is low.CXCL12 were not detected in all three cell lines.2.Construction and identification of CXCR4⁺or CXCR4^-NSCLC cell2.1 Construction and identification of CXCR4 low-expression NSCLC cellsAfter lentivirus transfection and puromycin selection,NCI-H1299 cells were harvested and RT-PCR and western blot results showed that the CXCR4 mRNA and proteins were significantly decreased.These results indicated that CXCR4 low-expression NCI-H1299cell lines were successfully established with CXCR4 shRNA lentivirus vector.2.2 Construction and identification of CXCR4 overexpression NSCLC cellsAfter CXCR4 overexpression with lentivirus,the CXCR4 mRNA level and protein in NSCLC cell were significantly elevated.This results showed that the CXCR4overexpression cell lines were established.2.3.Effects of CXCR4 on NSCLC proliferation.With the CCK-8 kit,CXCR4 inhibition resulted in NCI-H1299 lower proliferation rate.However,overexpression of CXCR4 promoted the proliferation of H1975.These results showed that CXCR4 played important role in NSCLC proliferation.2.4 Effects of CXCR4 on NSCLC migration/invasion.Wound scratch assay and transwell migration chamber experiments showed that CXCR4 downregulation blocked the horizontal and vertical migration ability of NCI-H1299 cells.Meanwhile,overexpression of CXCR4 enhanced the migration of H1975 cells.Together,CXCR4 participates the NSCLC cell migration.3.Effects of CXCR4⁺NSCLC cell on osteoclastogenesis3.1 Effects of NCI-H1299 cells on RAW 264.7 monocyte cell differentiation into osteoclast.NCI-H1299 cell conditional medium could induce the expression of TRAP and CK expression in RAW264.7 cells.TRAP staining showed that RAW264.7 cells have a higher rate into osteoclast.After lower-expression of CXCR4 in NCI-H1299 cells,the ability of its CM to promote the differentiation of RAW264.7 to OCs was inhibited.3.2 Effects of H1975 cells on RAW 264.7 monocyte cell differentiation into osteoclast.H1975 cell conditional medium could induce the expression of TRAP and CK expression in RAW264.7 cells.TRAP staining showed that RAW264.7 cells have a higher rate into osteoclast.After over-expression of CXCR4 in H1975 cells,the ability of its CM to promote the differentiation of RAW264.7 to OCs was further enhanced3.3 Effects of CXCR4 antagonist AMD3100 on CXCL12-CXCR4 axis mediated osteoclastogenesisUnder the stimulation of CXR4-overexpression H1975 conditional medium,the addition of AMD3100 significantly blunted the expression of TRAP and CK expression in RAW264.7 cells compared to the control group.Likewise,TRAP staining showed that AMD3100 inhibited RAW 264.7 monocyte cell differentiation into osteoclast.4.Mechanism of CXCR4⁺NSCLC cell promoting osteoclastogenesis4.1 Expression of VCAM-1,VEGF and MMP-9 in CXCL12 induced H1975^-OEVCAM-1,VEGF and MMP-9 were measured with ELISA kits.All of the above genes were significantly upregulated of which the fold of VCAM-1 change was the upmost one.The mRNA levels changes were similar.However,the VCAM-1 mRNA levels did not change significantly.4.2 CXCR4-CXCL12 axis activates downstream signaling including ADAM17To further investigate the regulation of sVCAM-1,we co-stimulated the NSCLCs with both CXCL12 and MMPs inhibitors TAPI-2 or TAP-I.a.ADAM17 was elevated in CXCR4-overexpressed H1975 cells.The treatment with either AMD3100 or TAPI-2 could block the ADAM elevation in CXCR4-overexpressed H1975 cells.b.Both AMD3100 and TAPI-2 could significantly decrease the sVCAM-1,VEGF and MMP-9 amount in the conditional medium from CXCR4-overexpressed H1975 cells.c.TAPI-1 significantly decreased the expression of ADAM17 in CXCR4-overexpressed H1975 cells.TAPI-1 also significantly decreased sVCAM-1 levels in the conditional medium from CXCR4-overexpressed H1975 cells.4.3 TAPI-1 and TAPI-2 could inhibit CXCL12-CXCR4 axis mediated osteoclasto genesisThe addition of TAPI-1 or TAPI-2 to the conditional medium from CXCR4-overexpressed H1975 cells decreased the TRAP and CK mRNA expression in RAW264.7cells.TRAP staining showed that TAPI-1 or TAPI-2 also attenuated RAW264.7 cells osteoclast differentiation induced by the conditional medium from CXCR4-overexpressed H1975 cells.ConclusionThis study investigated the expression of CXCR4 in the bone metastasis tissue of NSCLC and different NSCLC cell lines,and established CXCR4 overexpression cell lines.Further,we explore the mechanisms that CXCL12-CXCR4 axis participates NSCLC migration,proliferation and osteoclastogenesis.The conclusions are summarized as follows.1.CXCR4 expressed universally in the bone metastasis tissue of NSCLC and different NSCLC cells,especially in the area the bone was severely invaded,and the number of OCs is correlation with CXCR4.2.CXCR4-CXCL12 axis contributes to the proliferation and migration of NSCLC.3.NSCLC cells secrete VCAM-1,VEGF and MMP-9 through CXCR4-CXCL12 axis to promote osteoclastogenesis.Blockade of CXCR4-CXCL12 signaling or ADAM17inhibition could blunt NSCLC-induced osteoclastogenesis.To summarize,NSCLC enhanced osteoclastogenesis and formed bonemetastasis through CXCR4-CXCL12 axis and its downstream cytokines.