The Origins,Pathophysiological Roles And Regulatory Mechanisms Of 27-Hydroxucholesterol In Breast Cancer

Background and significance:Breast cancer is one of the most common tumors in women.It is reported that the abnormal cholesterol metabolism is an important risk factor for breast cancer.27-Hydroxycholesterol(27-HC)is one of the metabolites of cholesterol,which is identified as an endogenous selective estrogen receptor modulator(SERM)with the ability of promoting the growth of estrogen receptor(ER)-positive breast cancer cells.The clinical data reveals that there is elevated 27-HC content in breast tumors.The expression profiles of TCGA tumor samples and breast cancer cell lines reveals that the expression of CYP7B1,the oxysterol 7?-hydroxylase that degrades 27-HC,is significantly decreased in tumor tissues compared to the normal breast tissues.Some studies indicate that there is no direct connection between 27-HC accumulation in breast cancer and circulating 27-HC in blood.It is suggested that the elevated 27-HC content in tumor maybe originate from the local cholesterol metabolism.It is well known that there are a lot of macrophages in breast tumors,which promote the growth and migration of tumor cells.In this study,we are aiming to explore the origin,pathophysiological roles and regulatory mechanisms of 27-HC in breast cancer.Research design and methods:1.The expression of CYP27A1 and the secretion of 27-HC in breast cancer cells and monocyte/macropahge cells: 1)Western blots were applied to detect the expressions of CYP27A1 and CYP7B1 in human ER-positive breast cancer cell line MCF-7,ER-negative breast cancer cell line MDA-MB-231 and human monocytic leukemia cell line THP-1.2)Protein expression and immunofluorescence of CYP27A1 were performed in the co-cultured monocytes and breast cancer cells.3)The expressions of CYP27A1 in THP-1 and THP-1 treated by breast cancer media for 48 hrs were examined.4)Secretion of 27-HC in MCF-7,MDA-MB-231 and THP-1 treated with 100 nM PMA or not were compared by ELISA.5)Cell proliferation was evaluated by CyQuANT Cell Proliferation Assay Kit in MCF-7 with the stimulation of 27-HC.2.The expression and methylation of CYP7B1 in human breast cancer tissues: The expression and methylation of CYP7B1 were analyzed in samples of TCGA data base,related cell lines and clinic breast samples,and the expression of CYP7B1 in related breast cell lines were modulatedby the DNA de-methylatiing agent.3.Roles of tumor cells and their secretions to the differentiation of monocytes were analyzed: 1)Observation of the morphology of THP-1 when being co-cultured with breast cancer cells,MCF-7 or MDA-MB-231 for 48 hrs.2)HE staining and transwell culturing detection for observing the morphology of THP-1 after being stimulated by the conditional media from breast cancer cells for 48 hrs.3)The expressions of CD11 b and MMP-9 in THP-1 with or without stimulation of PMA or the conditional media were detected by western blot.4)The expression of CD14,CD25,CD163,IL-10 in THP-1 under stimulation of conditional media were analysed by Q-PCR.5)Flow cytometry to analyze the expression of membrane protein CD163 in THP-1 and THP-1 stimulated by conditional media of breast cancer cells.6)The expressions of CD86,TNF-?,IL-6,IL-1?,CD206,ARG1,PPAR?,IL-13 and IL-10 in mouse primary peritoneal macrophages were examined by Q-PCR under the stimulation of 4T1(mouse breast cancer cell line)conditional media.4.The effect of tumor derived exosomes on polarization of monocytes.1)The exosomes were collected from the meida of breast cancer cell cultures by ultrahigh-speed centrifugation and observed with transmission electron microscope.2)The expressions of CD11 b,MMP-9 were compared by western blot and the mRNA level of CD14,CD25,CD163,IL-10 were also examined by Q-PCR in the exosome-treated THP-1.5.The secretion of 27-HC in subtypes of macrophages were compared.1)The isolated mouse primary peritoneal macrophages were polarized to M1 type or M2 type by 200 ng/ml LPS or 10 ng/ml IL-4 for 48 h,respectively.2)The expression of CD206 and secretion of 27-HC were examined in M0,M1 and M2 type macrophages.6.The effect of 27-HC on macrophages was examined.The mRNA expressions of chemokines CCL2,CCL3,CCL4 were compared in THP-1 derived macrophages and mouse peritoneal macrophages under the incubation of 1 ?M 27-HC for 24 h.7.Preparation of 4T1 mouse tumor model.Divide the 8 weeks old female Balb/c mice into two groups.Inoc ulate 4T1 cells in the left abdomen,iniculated mixture of 4T1 with peritoneal macrophages from healthy mice(the first group)or mixture of 4T1 with peritoneal macrophages from tumor bearing mice(the second group)in the right abdomen,respectively.The bilateral tumor-bearing mice were sacrificed three weeks later for detection: 1)Blood routine;2)tumor weight;3)HE staining of the vital organs;4)Measurement of the infiltration of macrophages and contents of 27-HC in tumor tissues;5)RNA-seq analysis: Performed RNA-seq analyses in tumor tissues of six mice to look for the distinctly differentiated genes and analyze the relative pathway.Results:1.The expression of CYP27A1 was significantly increased in monocytes and their derived macrophages compared to breast cancer cells.2.The methylation of CYP7B1 was significantly increased in breast tumor tissues compred to normal breast tissues.3.Monocytes were likely differentiated to M2 type of macrophages,which produce more 27-HC,after being co-cultured with breast cancer cells or being exposed to breast cancer cell-derived exosomes.4.27-HC not only stimulated cancer cell proliferation,but also facilitated macrophages to express the chemokines CCL2,CCL3 and CCL4.5.There is a positive correlation between the content of 27-HC and quantity of M2 type macrophages in 4T1 derived mouse breast tumor tissues.6.Co-injection of peritoneal macrophages with 4T1 inhibit the infiltration of macrophages into tumor tissue might through leukocyte trans-endothelial migration pathway.Conclusions:1.The abundant infiltrated macrophages are the main source for elevated 27-HC in breast tumors.The highly expressing of CYP27A1 in macrophage increased the product of 27-HC;on the other side,hyper-methylation of CYP7B1 in breast cancer cells decreased the degradation of 27-HC.2.The monocytes were polarized to M2 type macrophages by the exosomes derived from breast cancer cells,which promoted the growth and metastasis of tumor cells through elevating 27-HC secretion.3.27-HC is a media for macrophage to modulate the tumor microenvironment.The 27-HC secreted by macrophage in tumor tissue promoted the cancer cell proliferation through paracrine way,and facilitated macrophages expressing the chemokines through autocrine way.