The Biological Function Of Thromboxane A₂ In Breast Cancer Lung Metastasis Of Mice And Dietary Intervention

The incidence and mortality of female breast cancer are still increasing all over the world,and tumor distal metastasis is the main cause of death.As an important part of tertiary prevention of cancers,how to reduce the risk of distal metastasis in breast cancer patients is a changeling clinical topic.Tumor metastasis is a multi-step biological process.Some common characteristics of tumor cells and secondary organs with metastatic potential have always been common strategies for studying the mechanism of distal metastasis of breast cancer and exploring potential prevention targets.Thromboxane A₂（TXA₂）,derived from the 20-carbon unsaturated fatty acid arachidonic acid,exists in various tissues and cells.It plays a signaling role through the corresponding G protein-coupled receptors（GPCRs）to mediate physiological and biochemical activities in the host organism.Previous studies revealed that thromboxane synthase（TBXAS1）is highly expressed in the distal metastatic organs of breast cancer patients.The recurrence and metastasis of cancer are often accompanied by a hypercoagulable state of blood embolism.It is known that TXA₂,as a vital small molecule metabolite,is involved in platelet aggregation and smooth muscle contraction,but its biological functions and mechanism of action in the distal metastasis of breast cancer remain unclear.This study focused on the scientific question of“whether and how TXA₂ pathway affects lung metastasis of breast cancer”.This study aimed to investigate the safety and effectiveness of TXA₂ pathway as a target for the prevention and treatment of cancer distal metastasis from the aspect of signal molecules by combining it with the classical“seed and soil”theory.The main research results are as follows:1.Firstly,the potential relationship of TXA₂-TP pathway in breast cancer metastasis was studied.BABL/c mice were selected to construct a 4T1 spontaneous lung metastasis model of breast cancer.Combined with clinicopathological data of breast cancer patients,sub-clonal cell lines were isolated from distal organs to analyze the potential pathological significance of TXA₂pathway and to identify the correlation between the abnormal activity of TXA₂ pathway and breast cancer metastasis.Here are the results:（1）Combined with the clinical data of TCGA breast cancer patients,the analysis results indicated that TP expression in tumor tissue was negatively correlated with the prognosis and survival of the diagnosed patients.（2）Based on the clinical samples,the analysis of common genes in lung,liver,bone,and brain organs with a high incidence of breast cancer metastasis showed there was an up-regulated expression of TXA₂ receptor TP.（3）The sub-clonal lines of mouse blood-circulating tumor cells were isolated and compared with the parent 4T1 cells,and it was found that there was an up-regulated expression of TXA₂ receptor TP.（4）It was found that the level of serum TXA₂ increased sharply by more than 6.5 times relative to normal mice,and that platelet aggregation was significantly enhanced（p<0.001）.2.Secondly,the importance and necessity of TXA₂-TP pathway in lung metastasis of breast cancer were evaluated through the construction of a spontaneous lung metastasis model of TP gene silencing cells and gene defect mice and by combining with pharmacological means.Here are the results:（1）Compared to the Mock-4T1 cell group,the injection of si TP-4T1 cell line into mouse breast fat pad effectively prolonged the survival time of mice with tumor,reduced the number of lung metastases,and inhibited the growth rate of tumor formation at the primary site.（2）In the host,the loss of the TP gene significantly inhibited the formation of lung metastasis of breast cancer,thus prolonging the survival of mice.（3）TXA₂ biosynthesis inhibitor aspirin was taken orally at low and medium doses（about 60-210 mg daily）.It was found that the serum TXA₂ level of mice was reduced by more than 95%,the formation of lung metastases was significantly reduced,and the survival period of mice was prolonged.Importantly,it was found serum TXA₂ levels were positively correlated with the number of lung metastases.Hence,TXA₂-TP pathway is an important factor mediating lung metastasis of breast cancer in mice.3.Based on the above correlation and functional conclusions,the key link and molecular mechanism of TXA₂-TP pathway in the distal metastasis of breast cancer were analyzed by cell tests.Besides,the signaling pathway and mode of action of TXA₂-TP mediated breast cancer anoikis resistance phenotype were clarified.The blood survival mode of circulating tumor cells is simulated by using a cell suspension culture model.Here are the results:（1）The malignant characteristics of breast cancer cells（e.g.,non-anchorage-dependent growth and invasion ability）were evaluated,and it was found that the loss of the TP gene in the cells inhibited malignant transformation.（2）Inhibiting the TXA2-TP pathway significantly reduced the number of circulating tumor cells in mice.（3）TXA₂,as a growth factor,could enhance the phosphorylation degree of PI3K/AKT when cells detached matrix attachment,providing survival signals for breast cancer cells with anoikis resistance.Warman penicillin（PI3K inhibitor）could block the influence of TXA₂ on AKT pathway.（4）TXA₂ as a signaling molecule,promoted survival in the way of TP-dependence.4.Finally,the above findings were applied to the targeted screening of dietary function factors to evaluate the possibility of targeting TXA₂-TP pathway for the prevention and treatment of distal metastasis of breast cancer and also lay a foundation for the wider application of dietary supplements.Dietary flavonoids were screened from the daily diet by using the feature of breast cancer cell anoikis resistance and the target of TXA₂-TP signaling axis,and then the possibility of dietary supplement assisting/preventing breast cancer metastasis was discussed.Here are the results:（1）Based on 7 kinds of flavonoids from food sources,it was found that apigenin had the most significant effect on inhibiting TXA₂ synthesis and inducing breast cancer cell anoikis,second only to aspirin.（2）The safety and effectiveness of apigenin intake were evaluated by using a spontaneous lung metastasis model of mouse breast cancer.It was found that 25mg/kg and 50mg/kg daily intake could reduce TXA₂ levels in plasma and inhibit the occurrence and development of lung metastases in mice,without producing any effect on the body weight,organ index,and blood cell index of mice.In conclusion,this study finds TXA₂-TP pathway mediates breast cancer cell anoikis resistance for the first time.It is clarified that TXA₂,as a signaling molecule-dependent receptor TP,maintains the continuous activation of PI3K/AKT pathway when breast cancer cells detach stromal adhesion,thus mediating the occurrence and development of distal breast cancer metastasis.Dietary plant compound apigenin,as a chemical agent targeting TXA₂ biosynthesis,can promote breast cancer cell anoikis resistance and inhibit the occurrence of lung metastasis of breast cancer in mice.Therefore,TXA₂-TP can be used as a new target for the treatment/prevention of distal metastasis in breast cancer patients.In addition,it also provides a scientific basis and theoretical basis for the directional development of dietary supplements and drugs in the future and the comprehensive prevention and treatment of breast cancer.