RAGE/sRAGE Promotes Bone Metastasis In Non-small Cell Lung Cancer By Regulating The Pre-metastatic Niche

Background and ObjectivesLung cancer is the main malignant tumor threatening the human health.There are2,206,771 new cancer diagnoses and 1,796,144 cancer-related deaths in the world every year,with non-small cell lung cancer(NSCLC)accounting for 85% of of them.Bone is one of the most common metastatic sites in NSCLC,and 30-40% of NSCLC patients suffer from bone metastasis.The consequence of bone metastasis is often devastating.On the one hand,bone metastasis accelerates the death process of NSCLC patients,with the median survival time of only 6-8 months after metastasizing to bone.On the other hand,bone metastasis causes skeletal-related events which seriously reduce the patient's quality of life.Currently,the clinical curative effect for NSCLC bone metastasis is unsatisfactory.Therefore,it is of great importance to explore the mechanism of NSCLC bone metastasis at the molecular level and to find the key genes regulating bone metastasis in NSCLC.NSCLC bone metastasis is a tightly regulated multi-step process.According to the hypothesis of “seed and soil”,tumor cells(seeds)metastasize to specific organ site(soil)depends on the cross-talk between seeds and soil rather than a random process.Actually,primary tumors could “prepare” the local microenvironment of distant organs for cancer cell colonization before their arrival.NSCLC cells could secrete tumour-shed extracellular vesicles and tumour-secreted factors that enable the bone microenvironment to encourage the outgrowth of incoming cancer cells.This favorable microenvironment created by primary tumor is called pre-metastatic niche.As an early event,the formation of pre-metastatic niche plays an important role in NSCLC bone metastasis,and blocking the formation of pre-metastatic niche has the potential to reduce the occurrence of bone metastasis from the very beginning.Therefore,this project aims to screen the key gene regulating bone metastasis of NSCLC by microarray analysis,and explore its regulatory role and mechanism in the bone metastasis,especially focusing on its role in the formation of pre-metastatic niche,so as to provide new therapeutic targets for NSCLC bone metastasis.Method1.Firstly,microarray analysis was conducted based on 10 tissue samples of NSCLC primary tumor and 10 samples of NSCLC bone metastasis to screen the differentiation expressed genes.Through bioinformatics analysis and literature review,RAGE was identified to be the key gene that might play an important role in bone metastasis of NSCLC.Next,we verified the correlation between expression level of RAGE and NSCLC bone metastasis at cell,tissue and patient levels.q RT-PCR and Western blot were conducted to compare the expression level of RAGE between A549 and A549-L6,a NSCLC cell with high tendency of bone metastasis.Immunohistochemistry assay was used to determine the differential expression of RAGE between primary NSCLC and bone metastasis.Survival analysis was performed to identify the relationship between RAGE expression and bone metastasis in NSCLC patients via using information in public database.2.Next,we explored the role of RAGE and its soluble form s RAGE in bone metastasis of NSCLC from two aspects: seeds and soil.A549 and H1299 stable cell strains with knockdown of RAGE were constructed.MTS and Transwell experiments were performed to explored whether RAGE promotes the proliferation and invasion of NSCLC cells(seeds).Experiments of pre-osteoblasts recruitment and differentiation were used to clarify the role of s RAGE in the transformation of bone microenvironment(soil).Adhesion,clone formation and MTS experiments were conducted to explore whether the promotion effects of osteogenic cells(soil)on the adhesion,colonization and proliferation of NSCLC cells(seeds)could be enhanced by s RAGE.Moreover,pre-metastatic niche model and tumor metastasis model by left ventricular injection were established to determine whether s RAGE promotes bone metastasis of NSCLC by regulating the pre-metastatic niche in vivo.3.In terms of mechanism research,secretory protein microarray was performed to identify the most obvious secretory protein differentially expressed in osteogenic cells after s RAGE treatment.KEGG and GO analyses were used to enrich the relevant signaling pathway.q RT-PCR,Western Blot and immunofluorescence experiments were performed to verify the differentially expressed protein and the signaling pathway.Osteoblast differentiation,MTS,clone formation and their rescue experiments were used to further determine the role of s RAGE in promoting the colonization and proliferation of NSCLC cells in bone microenvironment by regulating osteogenic cells.Results1.Firstly,RAGE was screened as a potential key gene of NSCLC bone metastasis by microarray analysis.q RT-PCR and Western blot showed that the expression level of RAGE in A549-L6 was significantly higher than that in A549.Immunohistochemistry showed that the expression level of RAGE in bone metastasis of NSCLC was much higher than that in primary tumor.Survival analysis showed that NSCLC patients with high expression of RAGE had significantly shorter bone metastasis-free survival,first progression survival and post progression survival than patients with low expression of RAGE.Therefore,RAGE was confirmed to be a bone metastasis-related gene.2.Next,MTS and Transwell experiments showed that the proliferation and invasion of NSCLC cells were slightly decreased after RAGE knockdown.Experiments of pre-osteoblasts recruitment and differentiation showed that s RAGE could transform the bone microenvironment by promoting pre-osteoblasts recruitment and differentiation.Adhesion,colony formation and MTS experiments showed that the promotion effect of osteoblastic cells on the adhesion,colonization and proliferation of NSCLC cells was enhanced by s RAGE pretreatment.Animal experiments showed that mice pretreated with s RAGE were more likely to develop bone metastasis of NSCLC.3.Secretory protein microarray and bioinformatics analysis showed that the content PDGF-C had the highest increase after s RAGE treatment,and the differential proteins were mainly enriched in TGF-? signaling pathway.q RT-PCR and Western blot analysis confirmed that the expression of PDGF-C was significantly increased after s RAGE treatment.Western blot and immunofluorescence experiments showed that s RAGE activated TGF-?/Smad signaling pathway in osteogenic cells.Osteoblast differentiation,MTS,clone formation and their rescue experiments further confirmed the role of s RAGE in promoting the colonization and proliferation of NSCLC cells in bone microenvironment by regulating osteogenic cells.ConclusionsBased on the above findings,we concluded that RAGE is a key gene regulating bone metastasis of NSCLC.NSCLC cells with high expression of RAGE could “prepare”pre-metastatic bone microenvironment by secreting s RAGE.s RAGE recruits osteoblast precursors in the bone microenvironment and promotes their differentiation into osteoblasts.At the same time,s RAGE promotes the secretion of growth factor PDGF-C by osteogenic cells via TGF-?/Smad signaling pathway.As a result,an osteogenic pre-metastatic niche was formed where the microenvironment is favorable for colonization,proliferation and metastasis of NSCLC cells.