Adipocyte Microenvironment Promotes Metastasis Through Regulating PLOD2-mediating Metabolic Reprogramming In TNBC

Background: Breast cancer is one of the most common malignant tumors in women in China.Triple negative breast cancer（TNBC）is considered to be the most aggressive,metastatic subtypes of breast cancer.It has poorer prognosis than other subtypes of breast cancer.Triple negative breast cancer is characterized by the absence of specific molecular targets.And targeted therapy and endocrine therapy are ineffective for triple negative breast cancer.In the mammary gland,the breast epithelium is surrounded by adipose tissue.These adipocytes adjacent to the cancer cells are gradually dedifferentiated adipocytes to pre-adipocytes and constitute for tumor microenvironment.Lysine hydroxylase 2（PLOD2）is an important molecule of collagen cross-linking,which can participate in the formation of extracellular matrix.The purpose of this experiment is to explore the effect of PLOD2 on the migration and invasion of tumor cells in the triple negative breast cancer,and further to analyze the potential mechanism of PLOD2 in regulating the migration of triple negative breast cancer in order to provide a new perspective for the treatment of triple negative breast cancer.Methods: The differentially expression of PLOD2 in various tumors were identified from TCGA database.3T3-L1 cells were induced into mature adipocytes and cocultured with triple negative breast cancer cells for 3 days.Cell migration assay and invasion assay were used to detect the effects of tumor microenvironment on migration and invasive of breast cancer cells.Next,knock-down of PLOD2 using si RNA had effect on oxidative phosphorylation and glycolysis metabolism in triple negative breast cancer by using seahorse,and verified the correlation between PLOD2 and glycolysis enzyme expression by Western blotting and real-time fluorescence quantitative PCR.We further analyzed whether PLOD2 could affect the migration ability of tumor cells by regulating glycolysis metabolism and the specific mechanism of PLOD2 regulating glycolysis.Results: TCGA database showed differences in PLOD2 expression in breast cancer,and its expression level increased significantly in triple negative breast cancer.We found the expression of PLOD2 was significantly upregulated in vitro coculture system.In addition,we demonstrated migration and invasive of TNBC cells were increased after co-cultured with mature adipocytes.It was showed that knockdown of PLOD2 in TNBC cells inhibited the oxidative phosphorylation and glycolysis metabolism levels.Inversely,overexpression of PLOD2 in TNBC induced oxidative phosphorylation and glycolysis metabolism of TNBC cells.The correlation between the expression of PLOD2 and glycolytic enzymes were analyzed by TCGA.It was showed that PLOD2 was strongly correlated with glycolysis key enzymes,which including PGK1,LDHA,PGAM1 and PFKP.These results were further verified by protein immunoblotting and real-time fluorescence quantitative PCR analysis of the expression of glycolysis related enzymes in TNBC cells after knockdown and overexpression of PLOD2.2-DG,a glycolysis inhibitor,was used to suppress glycolysis metabolism in TNBC cells after knockdown and overexpression of PLOD2.The results showed that 2-DG inhibited the migration of negative control cells and PLOD2 overexpression cells,but 2-DG failed to increase the anti-migratory ability of PLOD2 silencing cells.Using Jasper database to explore the transcription factors involved in regulating glycolysis,and it was showed that the transcription factor STAT1,STAT2 and STAT3 that could regulate glycolytic key enzyme.Furthermore,it was showed that silencing of PLOD2 led to the inhibition of STAT1,STAT2,STAT3 expression,while PLOD2 overexpression resulted in the upregulation of STAT1 and STAT3 in TNBC cells.Conclusions: Adipocyte microenvironment promoted the abnormal expression of PLOD2 in TNBC cells.Migration and invasive abilities of TNBC cells were enhanced by adipocytes.Mechanistically,adipocytes induced the expression of PLOD2 in TNBC cells.PLOD2 could promote TNBC cells metastasis by modulating glycolysis metabolism in TNBC cells.