Mechanism Of Prostate Cancer Bone Metastasis Regulated By Host-derived CCL2 And Screening Of Potential Metastasis-related Markers

Part 1 Host-derived CCL2 or CCR2 on prostate cancer bone metastasisInteraction between host bone microenvironment and prostate cancer cells increases the expression and secretion of some important cytokines,chemokines and adhesion molecules,which promotes tumor cell growth in bone and results in bone lesions.CCL2 is not only secreted by prostate cancer cells,but also produced by cells in the bone microenvironment in large quantities.Previous studies focused on the role of tumor-derived CCL2 on tumor growth and metastasis,and it is still unclear of the molecular mechanism of the host-derived CCL2 or CCR2 on prostate cancer-induced bone destruction.Our study first focused on the establishment of a specific animal model of bone metastasis of prostate cancer,which laid a solid foundation for studying the role and molecular mechanism of host-derived CCL2 in prostate cancer bone metastasis.To understand the role of host-derived CCL2 or CCR2 on prostate cancer-induced bone destruction,specific prostate cancer bone metastaticcells were intra-cardially injected into CCL2 knockout or CCR2 knockout mice.Metastatic bone lesion development was monitored and analyzed by BLI image.The results showed that a loss of host-derived CCL2 could significantly reduce the bone metastasis of tumor cells.Also,the proliferation of tumor cells was slowed down in bone.Moreover,the survival time of mice was significantly prolonged,and the osteolytic lesions of femur was significantly reduced.However,a loss of host-derived CCR2 increased the incidence of bone metastases in prostate cancer,and the survival time of mice was significantly shortened.Part 2 Mechanism of a loss of host-derived CCL2 in reducing bone metastasis of prostate cancerTumor cells in the bone marrow colonization and growth requires several steps,including the formation of blood vessels,local metastasis and invasion,from the primary tumor into the blood vessels,and then overflow from the blood vessels,adaptation,survival and colonization in the bone marrow.Tumor cell-derived CCL2 can bind to CCR2 to promote tumor proliferation and angiogenesis,as well as to recruit mesenchymal cells for tumor metastasis.The main purpose of this study was to investigate the role of host-derived CCL2 in bone metastasis of prostate cancer and try to explain its mechanism on bone metastasis in prostate cancer from different aspects.Based on the pre-established model of prostate cancer bone metastasis,we first isolated bone metastatic cells from WT mice or CCL2 KO mice and mRNAs expressed in different groups were analyzed by RNA-Seq.Secondly,we identified CCL2 related gene CCND2 by comparing bone metastatic cells derived fromWT mice and CCL2 KO mice.Thirdly,the number of MDSC cells in bone microenvironment were detected by flow cytometry.Finally,the molecular mechanisms of bone resorption and bone formation caused by host-derived CCL2 were analyzed in vivo and in vitro.The results were as follows:(1)We identified the biological characteristics of prostate cancer bone metastasis cells;(2)Through the analysis of bone metastatic cells of CCL2 KO mice and bone metastasis of WT mice,we identified the relationship between CCND2 and CCL2 by KEGG pathways.qRT-PCR also confirmed that the CCND2 gene in bone metastatic cells isolated from CCL2 KO mice was significantly lower than that of WT.The bone metastatic cells isolated from CCL2 KO mice also showed significantly less proliferative ability than that in parental cells.(3)A loss of host-derived CCL2 reduced the production of MDSCs in prostate cancer bone metastasis microenvironment and reduced the bone lesions of prostate cancer,including reducing the number of Trap positive cells and PCNA positive cells in femur and increasing the bone mineral density;(4)It was confirmed in vitro that the ability of osteoclast differentiation was weakened in CCL2 knocked-out cells treated with tumor cell conditioned media,and the expression of marker NFATc1 in osteoclasts was also significantly decreased by qRT-PCR analysis.Part 3 Screening of potential metastatic markers for prostate cancerProstate cancer is the most common malignancy in men in the United States.Most prostate cancers have been diagnosed with advanced tumors or metastases.Skeletal bone is the most common metastatic site ofprostate cancer,and bone metastases are the leading cause of high mortality.In this section,we used two methods to screen prostate cancer metastasis-related biomarkers.Firstly,parental cells and bone metastasis cell were performed to RNA-Seq and the up-regulated and down-regulated mRNAs were performed with Gene ontology(GO)and KEGG pathway analyses separately to identify potential metastatic markers of prostate cancer.Secondly,we used the Oncomine,cBioPortal,and TCGA public databases to compare differentially expressed mRNAs between prostate cancer and paracancerous tissues,and to investigate the potential correlation between gene abnormalities and clinical features in PCa to evaluate disease progression and prognosis.We found that C10orf116 loss may not only contribute to an understating of the invasion and metastasis ability of PCa but also evaluate the prognosis of patients.Conclusion1.A loss of host-derived CCL2 slowed down the growth of prostate cancer in the bone,whereas a loss of host-derived CCR2 increased the incidence of bone metastases in prostate cancer.2.A loss of host derived of CCL2 may promote the immune response by suppressing the recruitment of MDSC cells to the tumor microenvironment and inhibit the growth and colonization of tumor cells in the bone.3.A loss of host derived of CCL2 led to osteoclast differentiation disorder and inhibited osteolytic bone metastases in prostate cancer.4.We screened some biomarkers that may be related to the bone metastasis of prostate cancer by analyzing RNA-Seq data and carried outpreliminary identification,and the follow-up function experiments are still on going.5.We found that C10orf116 loss may not only contribute to an understating of the invasion and metastasis ability of PCa,but also evaluate the prognosis of patients by mining public database data.