The Regulation Role And Mechanism Study Of CCL7/CCR3 Pathway In Non-small Cell Lung Cancer Bone Metastasis

Background and objectiveLung cancer is the second highest incidence of malignant tumors,and the mortality rate in men and women are currently ranked in the top second.As of 2014,the number of lung cancer cases in the United States has reached 430090,with an annual growth rate of 224210.In recent years,along with the aggravation of the aging of the population and environmental pollution in our country,the incidence of lung cancer also began to increase exponentially.There are about 400 thousand people are diagnosed with lung cancer each year in China,with the incidence rate of 61.4/10 million,becoming the largest population of lung cancer in the world.Although the diagnosis,surgery and chemotherapy have been greatly improved in the past ten years,the metastasis of lung cancer and mortality still remain high,and the 5 year survival rate is only about 15%.According to the pathological type,lung cancer can be classified into two major types: non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).The NSCLC accounts for about 87% of all lung cancers.The NSCLC is divided into three types: adenocarcinoma,squamous cell carcinoma and large cell lung cancer.The adenocarcinoma is the most common type of lung cancer.At present,most of the patients are initially diagnosed with advanced stage of lung cancer,and most of them lose the chance of operation.Even patients with lung cancer can be diagnosed early and underwent surgery,the rate of postoperative recurrence and metastasis is as high as 1/3.Tumor metastasis is an important cause of death.The NSCLC is commonly associated with m?ltiorgan metastasis leading to increased mortality.Bone metastasis represents one of the most deleterious clinical consequences of NSCLC.And 39% of patients with NSCLC have bone metastasis.The bone metastasis of NSCLC is mainly manifested as the destruction of bone,leading to fractures,severe bone pain and even paralysis,which seriously affects the quality of life and prognosis of patients.Our previous study found that the 3 year survival rate of the NSCLC patients with bone metastases was less than 30%,which is similar to previous findings.The bone metastasis of tumor is a complex process,and the mechanism of bone metastasis of lung cancer is mainly considered as follows: First,The epithelial mesenchymal transition of tumor cells and the lesion was fallen away from the primary NSCLC entering the blood circulation.Second,The tumor cells were transferred to bone tissue with blood circulation.Finally,the tumor cells promote bone destruction and tumor proliferation through the interaction between osteoblasts and osteoclasts,resulting in bone metastases.Chemokine ligand,playing an important role in the biological characteristics of tumor and tumor immunity,is a small molecule chemical chemotactic factor of less than 100 amino acids.According to the difference of two cysteine residues of Chemokine ligand,it can be divided into three types: CC,CXC and XC.CCL7 is a member of the CC chemokine family,and it has been found to be highly expressed in breast cancer,renal cell carcinoma,gastric cancer and colorectal cancer,promoting the proliferation,migration and invasion of tumor.Meanwhile,CCL7 also plays an important part in the immune system,which can regulate the immune cells such as monocytes,T cells,NK cells,dendritic cells and so on.However,the role and mechanism of CCL7 in lung cancer is still unclear.Research contents and methodsWe collected 93 cases of primary tumors and para-tumor lung tissue specimens and 49 cases of bone metastasis specimens from patients with NSCLC who were surgical treated at the Changzheng Hospital Affiliated to the Second Military Medical University.We used microarray to detect the difference of gene expression between NSCLC primary and bone metastases.qRT-PCR and IHC staining were also used to detect the expression difference of CCL7 in para-tumor lung tissue,NSCLC primary tumor and NSCLC bone metastases.We analyzed the relationship between CCL7 expression and prognosis in patients with NSCLC by IHC staining.Then,we used CCL7 to stimulate NSCLC cell line A549 and PC9,and examined the effect of CCL7 on proliferation,migration,invasion,EMT and differentiation of osteoclast induced by NSCLCThe main receptors of CCL7 include CCR1,CCR2 and CCR3.We used qRT-PCR and IHC staining to detect CCR1,CCR2 and CCR3 in the differential expression of NSCLC in primary and metastatic bone.In order to understand the inhibitory effect of CCR3 on CCR2 and CCL7 in NSCLC cells by using CCR2 inhibitors and CCR3 inhibitors.We further constructed a A549-shCCR3 stable cell line of CCR3 inhibition by lentiviral transfection and examined the inhibitory effect of CCR3 on the metastatic ability of A549 cells in nude mice.We detected the changes of gene expression in CCL7 stimulated A549 cells by sequencing the gene expression profiles.At the same time,we explored the possible downstream pathways through GO analysis and KEGG analysis,and verified the regulatory role of CCL7/CCR3 pathway on downstream gene expression by qRT-PCR.ResultsWe found that the expression of CCL7 was significantly higher in NSCLC bone metastases than in the primary NSCLC,and the clinical prognosis of patients with high CCL7 expression in NSCLC was significantly lower than that in patients with low expression of NSCLC.After stimulation of A549 and PC9 cells by CCL7,we found that CCL7 could promote the migration and invasion of A549 and PC9 cells,but had no effect on cell proliferation,and we found that CCL7 could promote the EMT of A549 and PC9 cells by Western blot as well.After stimulation of BMM cells by CCL7,we found that CCL7 could not directly promote osteoclast differentiation.But the condition media from A549 cells stimulated with CCL7 showed a more significant effect in the osteoclast differentiation of BMM cells than the condition media from control A549 cells.CCR2 and CCR3 were found to be highly expressed in NSCLC bone metastases relative to the primary tumor,and were associated with overall prognosis and tumor progression in patients with NSCLC.In addition,CCL7 can promote the expression of CCR3.Duing to the inhibition of CCR2 and CCR3 by inhibitors respectively,we found that inhibition of CCR3 could inhibit the migration,invasion,EMT and the regulation of osteoclast differentiation of CCL7 on A549 and PC9 cells.However,the inhibitory effect of CCR2 on CCL7 in A549 and PC9 cells was not significantly affected.We constructed the A549-shCCR3 stable cell line by lentivirus transfer to the left ventricle of nude mice.Then,we found that the inhibition of CCR3 in nude mice could significantly inhibit the formation of A549 cells in vivo imaging.We found that CCL7 can promote the expression of several genes related to tumor development,such as IL1 B,CYP1B1 and FLCN and so on,by using the analysis of gene expression profiling and Bioinformatics.We also found that CCL7 may play a role in activating the Ras pathway.We verified the promotion effect of CCL7 on the expression of these downstream genes in A549 and PC9 cells by qRT-PCR,and found that inhibition of CCR3 could inhibit the regulation of CCL7 on the downstream gene expression to a certain extent.ConclusionBased on the above results,we suggest that CCL7 may play an important role in the process of NSCLC metastasis,especially bone metastasis.CCL7 can promote the migration and invasion of NSCLC cells by inducing the EMT of NSCLC cells.CCL7 can also promote the differentiation of osteoclast induced by NSCLC cells.CCL7 plays a role in activating CCR3 in NSCLC cells,while inhibition of CCR3 in vivo can significantly inhibit the transfer of NSCLC.In addition,CCL7/CCR3 pathway may function in NSCLC through the activation of Ras signaling pathway.These findings suggest that the CCL7/CCR3 pathway may play an important role in bone metastasis of NSCLC,and may serve as a novel therapeutic target for NSCLC bone metastasis.