Sema4D Expression And Secretion Induced By HIF-1? To Inhibits Osteogenesis And Participates In Bone Metastasis Of Lung Cancer

Objectives:Lung cancer is the most common malignant tumor in China with the highest morbidity and mortality.Bone metastasis is one of the frequent complications of advanced lung cancer,which can lead to bone-related events such as severe pathological fractures,malignant pain,hypercalcemia and spinal cord compression,and seriously affect the quality of life and survival time of lung cancer patients.More than 86%of bone metastases of lung cancer are characterized as osteolytic bone destruction.The main mechanism is that when lung cancer cells metastasizes to the bone,they will destroys the bone remodeling composed of osteoblast-mediated osteogenic and osteoclast-mediated osteolytic,and ultimately leads to osteolytic bone destruction under the action of various tumor factors,bone matrix growth factors and microenvironmental factors?such as hypoxia?,but its specific mechanism has not been fully elucidated.Since osteoclasts are the only cell known to be involved in bone resorption,their roles and mechanisms in osteolytic bone destruction have been extensively elucidated.At present,many anti-osteolytic drugs such as bisphosphonate and denizumab targets for inhibiting osteoclasts and their mediated bone resorption,which have been approved for clinical use in the past,are therapeutic.However,the clinical efficacy of the existing treatment regimen is still limited.It is urgent to clarify more detail molecular mechanism of osteolytic bone destruction induce by tumor bone metastasis to provide a new target for the prevention and treatment of tumor bone metastasis.Recent studies have confirmed that osteoblasts also play a critical role in the development of tumor bone metastasis.Osteoblasts are the first responding cells after tumor bone metastasis,and functioned in regulating the migration and differentiation of osteoclasts and promoting tumor progression by releasing a large number of osteoclasts and tumor regulatory factors such as RANKL/OPG,M-CSF,Semaphorin 3A?Sema3A?,VEGFA,etc.Previous studies have confirmed that the osteogenic capacity of osteoblasts can be significantly inhibited in tumor bone metastasis.Lung cancer cells can inhibit osteoblast-mediated osteogenic differentiation has been confirmed by our group,but the mechanism is unclear.Clarify its mechanism can provide a new breakthrough for the prevention and treatment of osteolytic bone destruction in bone metastasis of lung cancer.Hypoxia is an important feature of the bone microenvironment,after tumor bone metastasis can lead to more severe hypoxia conditions due to the increase in oxygen demand and microvascular destruction of rapid tumor proliferation.Previous studies have demonstrated that tumor-associated hypoxia-mediated hypoxia-inducible factors?HIFs?are highly expressed in tumor bone metastases,and promote osteolytic progress of bone metastasis by inhibiting osteoblast differentiation and promoting osteoclastogenesis.Knocking out of HIFs in cancer cells can significantly reduce the degree of osteolytic bone destruction and increase bone mass,but the precise mechanism remains to be elucidated.Sema4D is a novel bone metabolism regulatory molecule recently been discovered.In normal bone remodeling,Sema4D expressed by mature osteoclasts and mediates osteogenic inhibition.Feedback of Sema4D provides a basis for inhibiting the function of osteoblasts to ensure the completion of the osteolytic step.Subsequent studies have shown that Sema4D plays an important role in osteoporotic diseases such as osteoporosis,multiple sclerosis,and acetabular dysplasia.Moreover,Sema4D also play mutiple roles in various physiological and pathological processes such as tumor progression,immune regulati on,angiogenesis,and regulation of bone metabolism.Additionally,in solid tumors such as oral squamous cell carcinoma and ovarian epithelial cancer,hypoxia regulates Sema4D expression through HIFs to control the endothelial cell migration and tumor vascular distribution,and tumor growth and.Therefore,we suspect that in lung cancer bone metastasis,hypoxia may regulate the expression and secretion of Sema4D in lung cancer cells through HIFs,mediate osteogenic inhibition and participate in osteolytic bone destruction mediated by bone metastasis of lung cancer.This study intends to elucidate the role of HIFs on the expression and secretion of Sema4D in lung cancer cells and its potential role in osteolytic bone destruction of lung cancer bone metastases by a series of experimental methods,and provides new targets and ideas for the prevention and treatment of bone destruction of lung cancer bone metastasis.Methods:1.The expression of Sema4D in lung cancer cell lines was detected by q-PCR and WB.The conditioned medium?CM?of lung cancer cells were collected and the secretion of Sema4D in lung cancer CM was detected by ELISA.Lung cancer conditioned medium were co-cultured with osteoblasts,and alkaline phosphatase staining and alizarin red S staining,expression of osteogenic differentiation genes?ALPL,Oxterix,Coll?1?were used to detect the osteogenic differentiation levels.Correlation between Sema4D expressi on in lung cancer cells and their osteogenic inhibition ability was anaylyzed.Sema4D shRNA transfection were used to interfered with the expression and secretion of Sema4D in lung cancer cells.To confirmed the role of Sema4D expression on the osteogenic differentiation inhibition of lung cancer cell CM,the conditioned medium from lung cancer cells with or without Sema4D interference was obtained and co-cultured with osteoblasts.The osteogenic differentiation levels were measured by the detection of osteogenic differentiation genes?ALPL,Oxterix,Coll?1?,alkaline phosphatase staining and alizarin red S staining;2.Hypoxia model was established by pre-treatment of lung cancer cells with CoCl₂?100?M?,conditioned medium from lung cancer under normoxia or hypoxia conditions were collected and co-culture with osteogenic precursor cells,osteogenic differentiation levels were measured by the detection of osteogenic differentiation genes?ALPL,Oxterix,Coll?1?,alkaline phosphatase staining and alizarin red staining,and the effect of hypoxia-induced lung cancer cell exocytokines induced by CoCl₂?100?M?on osteoblast differentiation was determined.3.The effect of hypoxia induced by 1%O₂ or CoCl₂?100?M?on the expression and secretion of Sema4D in lung cancer cells were detected by q-PCR,WB and ELISA.4.The effect of Sema4D shRNA transfection on the expression and secretion of Sema4D in lung cancer cells was detected by q-PCR,WB and ELISA.To determine the role of hypoxia-induced Sema4D expression and secretion in lung cancer induced osteoblast differentiation inhibition under hypoxia,Sema4D shRNA was transfected into lung cancer cells and pretreated with CoCl₂?100?M?,conditioned medium were collected as suggested and co-cultured with osteoblasts.Osteoblast differentiation levels was measured by alkaline phosphatase staining and alizarin red S staining,detection of osteogenic differentiation genes?ALPL,Oxterix,Coll?1?.5.The expression of HIF-1?and HIF-2?under hypoxia induced by 1%O₂ or CoCl₂?100?M?pretreatment were detected by WB and immunofluorescence.To determine the upstream regulatory mechanism of Sema4D,shRNA targeting HIF-1?or HIF-2?were transfection into lung cancer cells to interference the HIF-1?or HIF-2?expression,and the expression and secretion of Sema4D in lung cancer cells were detected.Dual luciferase assay was used to determine whether HIF-1?has direct regulated Sema4D expression and the detection site of Sema4D promoter region.The effect of conditioned medium of lung cancer cells with HIF-1?or HIF-2?knockdown on the differentiation of osteoblasts under hypoxic conditions were measured by alkaline phosphatase staining and alizarin red S staining,detection of osteogenic differentiation genes expression?ALPL,Oxterix,Coll?1?.6.The expression of ADAM17?a enzyme responsible for Sema4D cleavage?in lung cancer cells under hypoxia induced by 1%O₂ and CoCl₂?100?M?pretreatment were detected by WB and q-PCR.To determine the upstream regulatory mechanism of ADAM17,the effects of interference with HIF-1?or HIF-2?expression on the expression of ADAM17in lung cancer cells were observed.To defining the effect of ADAM17 on the secretion of Sema4D,after inhibiting the expression or function of ADAM17 in lung cancer cells by shRNA interference or adding ADAM17 specific inhibitor TAPI-1,the secretion level of Sema4D in conditioned medium from lung cancer induced by CoCl₂?100?M?was detected by ELISA.After silencing the expression of ADAM17 in lung cancer cells,the effect of conditioned medium from lung cancer cells under hypoxia on the osteoblast differentiation was measured by alkaline phosphatase staining and alizarin red S staining,and detection of osteogenic differentiation genes?ALPL,Oxterix,Coll?1?.7.Immunohistochemistry was used to detect the expression of HIF-1?,HIF-2?,Sema4D and ADAM17 in forty-nine cases of lung cancer bone metastases,and the correlation between their expression and location with clinical characteristics of patients including gender,age,number of invasive vertebral bodies,pathological tumor type,tumor differentiation and type of bone destruction were analyzed.The correlation between the expression of Sema4D,ADAM17 and HIF-1?,HIF-2?in bone metastases of lung cancer were analyzed.To determine the expression and clinical significance of HIF-1?,HIF-2?,Sema4D and ADAM17 in bone metastasis of lung cancer.Results:1.Sema4D is expressed and secreted by variety of lung cancer cell lines,and conditioned medium from lung cancer with higher Sema4D expression showed stronger inhibitory ability on osteogenic than that of lung cancer cell line with lower Sema4D expression.Silencing the expression and secretion of Sema4D in lung cancer cells by shRNA significantly reduced the in inhibition ability of lung cancer cell conditioned medium to osteoblast differentiation.2.Hypoxia induced by CoCl₂?100?M?pretreatment significantly promoted the effect of osteoblast differentiation inhibition by lung cancer cell conditioned medium.3.The expression of Sema4D mRNA and protein in lung cancer cells and its secretion in lung cancer conditioned medium were significantly improved by hypoxia.Konckdown of Sema4D expression lung cancer cell significnatly attenuates the inhibition ability on osteoblast differentiation by conditioned medium from lung cancer after CoCl₂?100?M?pretreatment.4.The expression and secretion of Sema4D in lung cancer cells were regulated by hypoxia in a HIF-1?but not HIF-2?-dependent manner.Double luciferase enzyme assay confirmed that HIF-1?directly binds to the 1771-798 base pair of the Sema4D promoter region and regulate its transcription.Silencing of HIF-1?but not HIF-2?significantly attenuated the inhibitory effect of CoCl₂-treated lung cancer conditioned medium on osteoblast differentiation.5.Hypoxia promotes the expression of ADAM17?enzyme for Sema4D cleavage?in lung cancer cells through HIF-1?but not HIF-2?-dependent manner.Inhibition of ADAM17 expression or function significantly reduced the secretion of Sema4D in CoCl₂-treated lung cancer cell conditioned medium.Silencing of ADAM17 expression in lung cancer cells significantly attenuated inhibitory effect of CoCl₂-treated lung cancer conditioned medium on osteoblast differentiation.6.Immunohistochemical results showed that HIF-1?,Sema4D and ADAM17 were highly expressed in osteolytic bone metastases of human lung cancer,and significantly correlated with poor tumor differentiation and osteolytic bone destruction.There was significnatly correlations between the expression of Sema4D,ADAM17 and HIF-1?in bone metastases of human lung cancer,but no correlation with HIF-2?expression was observed.Conclusions:In lung cancer bone metastasis,Sema4D is highly expressed in lung cancer cell lines and mediates the inhibition effect on osteoblast differentiation by lung cancer cells.Hypoxia promotes the expression of Sema4D in lung cancer cells directly through HIF-1?instead of HIF-2?-dependent manner,and regulates the secretion of Sema4D by regulating the expression of ADAM17,and mediates more aggressive inhibition on osteogenic differentiation induce by lung cancer under hypoxia.In the human lung cancer bone metastasis specimens,Sema4D,ADAM17 and HIF-1?were highly expressed in osteolytic bone metastasis and there was significant correlations were observed,which revealed the potential mechanism of osteolytic bone destruction in bone metastasis of lung cancer,and provided a new targets for the prevention and treatment of bone metastasis of related tumors.