Metabolic Reprogramming Promotes The Tamoxifen Resistance In ER-Positive Breast Cancer

Background: In recent years,the incidence of cancer and its death rate have continued to grow.Cancer has become a public health problem in China and even in the world.Among them,the incidence of breast cancer in women has always been high,which seriously threatens the women’s health.Breast cancer is divided into different subtypes based on molecular expression,of which more than 70% is estrogen receptor(ER)positive breast cancer.In the past few decades,tamoxifen has been the most important endocrine adjuvant for ERpositive breast cancer.However,unfortunately,many patients,including ER-positive breast cancer patients who respond to drugs at the initial stage of their medication,have tamoxifen resistance during treatment.There are different opinions about the possible mechanisms of drug resistance,and metabolic reprogramming of cells may be one of the important reasons for drug tolerance.The energy metabolism of cells affects the survival and development of cells,and the abnormal metabolism of tumor cells has drawn people’s great concern.Mitochondria are important sites for energy metabolism.The mitochondria of tumor cells often have functional abnormalities.Recent studies have confirmed that mitochondrial fusion/division may play an important role in the development of tumors.The maintenance of a dynamic mitochondrial network has an important effect on signal transduction and ATP production.Mitochondrial morphological transformation also involves cell survival,apoptosis and its metabolic homeostasis.However,whether mitochondrial fusion/fission is involved in tamoxifen resistance tumor cells is currently unclear.Purpose: To explore the differences in metabolic patterns and mitochondrial morphogenetic functions of tamoxifen-resistant cells,and to clarify the mitochondrial morphology and regulatory mechanisms of metabolic reprogramming in drug-resistant cells,providing important new ideas and new targets for effective monitoring and reversal of tamoxifen resistance.Methods: 1.Tamoxifen-resistant cell lines of ER-positive breast cancer cells MCF7 and T47 D were established.The cells were identified by STR sequencing analysis,cell morphology was analyzed by cell counter,and the biological function differences of the drug-resistant cell lines were detected by using MTT and plate colony formation assays.2.Parental and resistant cell lines were tested for glucose metabolism.The qPCR and Western Blot experiments were employed to screen important molecules that may be involved in the metabolic reprogramming of drug resistance.The 2-NBDG uptake assay was also used to detect the sugar uptake of the parental and drug resistant cells.The glucose detection kit was used to analyze the cell’s sugar utilization.The differences of glycolysis and oxidative phosphorylation in parental and resistant cells were also analyzed by XF extracellular flux analyzer.3.Observation of mitochondrial morphology and function.Membrane potential of the cells was detected by JC-1 staining.Mito-Tracker staining and transmission electron microscopy(TEM)were employed to detect the mitochondrial morphologies of the parental and drugresistant cells.ATP synthesis and reactive oxygen species(ROS)production were further analyzed for mitochondrial biological functions.4.The molecular mechanisms of mitochondrial fusion/fission involving in the reprogramming of drug resistance metabolism was examined.The qPCR and Western Blot experiments were used to screen the key molecules involved in cell resistance and their expressions of upstream related molecules in mitochondrial fusion/fission.Gene overexpression and interference experiments were performed to test the biological functions of obtained key molecules.Fluorescent reporter genes and Chip assay were further utilized to analyze its regulatory mechanisms.Results: 1.No significant difference in the sizes was found in the parental and resistant cells.The resistance of drug resistant cell lines to tamoxifen was also significantly enhanced.2.Both MCF7 and T47 D resistant cells showed enhanced sugar uptake and increased sugar utilization.The molecular enzymes involved in the glycolysis pathway were detected.Expression trends of related genes in MCF7/TR and T47 D resistant cells(T47D/TR)were slightly different.ECAR results showed that the MCF7/TR cells exhibited a higher glycolytic activity than the parental cells(MCF7/P),whereas there was no significant difference in the glycolysis of the T47D/TR and its parental T47D/P,except oxidation.Phosphorylated OCR was measured,and the OCR value in two cell lines were significantly higher than their parental cells.3.The membrane potentials of MCF7/TR and T47D/TR were higher than their parental cells.TEM and Mito-Tracker staining revealed that the mitochondrion of MCF7/TR cells was narrow and long,and the ridge structure was tighter.The ATP content of MCF7/TR and T47D/TR was higher than that of the parental cells,while the ROS content was significantly lower than that of the parental cells.4.The high expression of mitochondrial inner membrane fusion protein,optic atrophy protein 1(OPA1),in two resistant strains is closely related to the resistance of cell-resistant and drug-resistant cells.Interfering with the expression of OPA1 in drug-resistant strains increased the sensitivity of drug-resistant cells to tamoxifen,upregulated ROS levels,decreased OCR markers,and loose mitochondrial ridge structure.over-expressed OPA1 in the parental cells increased the tolerance of the cells to tamoxifen,decreased the ROS,increased the OCR index,and made the mitochondrial ridge structure more compact.The AKT-mTOR-P65 signaling axis was involved in the regulation of OPA1 expression.Conclusions: Activation of the AKT-mTOR-P65 signaling pathway upregulated the expression of OPA1,thereby altering the metabolic pattern of drug-resistant cells and promoting cell resistance to tamoxifen.