Research On The Mechanism Of MCAD Regulating Epithelial-mesenchymal Transition To Promote The Occurrence And Metastasis Of Triple-negative Breast Cancer

Breast cancer is one of the most common malignant tumors in women.In recent years,the incidence rate has continued to rise,and it has become the first disease of female malignant tumor incidence in China and even in the whole world,seriously threatening women’s health.Due to the high heterogeneity of breast cancer,different types of breast cancer have different risks and mechanisms of recurrence and metastasis.The triple negative breast cancer(TNBC)is a special type of breast cancer,which is characterized by strong invasiveness,high distant metastasis rate and poor prognosis.Estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)are not expressed in triple-negative breast cancer,which results in a lack of endocrine and anti-her2 treatment targets.Currently,there is no targeted standard treatment option for TNBC.It is important to study the mechanism of occurrence and distant metastasis of triple negative breast cancer in order to identify new potential target drugs.It has been showed that lipid metabolism is related to the occurrence and metastasis of tumor,and the current studies on tumor lipid metabolism mainly focuses on the de novo synthesis of fatty acids.Several studies have reported that the expression and activity of various enzymes involved in the fatty acid synthesis pathway in tumor cells are all up-regulated.Targeted inhibition of some enzymes(eg ATP citrate lyase,acetyl-CoA carboxylase,and fatty acid synthase or stearyl CoA desaturase)in the fatty acid synthesis pathway with small molecule inhibitors can cause tumor regression.However,these drugs have severely reduced the food intake and body mass of experimental mice,and restricted their application in anti-tumor therapy.Thereforc,the fatty acid oxidation pathway will become a new direction for anti-tumor treatment.In this study,we had analyzed more than 140 metabolites between two TNBC cells with different metastasis using metabolic profiling,the result showed that the fatty acidβ-oxidation metabolites in the high metastasis cell have increased,and medium chain acyl-CoA dehydrogenase(MCAD),the key enzymes catabolizing the first step of fatty acid β-oxidation,were significantly up-regulated in mRNA and protein level,suggesting that MCAD expression is closely related to metastasis of triple-negative breast cancer cells.To examine the role of MCAD in the progression and metastasis of TNBC,we used lentiviral infection technology to knock down and overexpress MCAD in breast cancer cells,we found that knockdown of MCAD gene expression in MDA-MB-231 and MDA-MB-468 cell lines significantly inhibited the cell proliferation,migration and invasion,and up-regulated the expression of epithelial genes such as E-cadherin but down-regulated the expression of mesenchymal genes such as Vimentin and N-cadherin,suggesting that the epithelial-mesenchymal transition(EMT)process was inhibited.However,ectopic expression of MCAD gene in MDA-MB-231 cells and non-triple negative breast cancer cells MCF7 cells significantly enhanced cell proliferation,migration and invasion,induced a mesenchymal genes expression but decreased epithelial gene expression and the cells showed an EMT phenotype.These findings suggest that MCAD can promote the proliferation,migration and invasion of triple negative breast cancer cells and induce the EMT process.More over,we use the GEPIA database to analyse the correlation between MCAD and EMT-related transcription factors,we found that the transcription factor Zinc Finger E-Box Binding Homeobox 1(ZEB1)has significant positive correlation with MCAD expression,and the expression of ZEB1 was consistent with expression of MCAD,both of which were highly expressed in MDA-MB-231 of triple-negative breast cancer cells.Knockdown of ZEB1 gene in MDA-MB-231 cells with ectopic expression of MCAD can largely inhibited MCAD-promoted proliferation migration and invasion proving that MCAD may affect breast cancer invasion and metastasis by regulating ZEB1.Further more,we evaluated the effects of MCAD over-expressing MDA-MB-231 cell line on breast cancer tumor growth and lung metastasis in vivo using mouse tumor xenograft models.The results showed that MCAD overexpression could significantly promote tumor growth and lung metastasis of mice,which was consistent with the results in vitro.In summary,this study systematically studied the effects of MCAD gene on the growth and metastasis of triple-negative breast cancer through in vitro and in vivo experiments.The results demonstrate that MCAD may promote the growth and metastasis of triple-negative breast cancer by regulating the transcription factor ZEB1 to induce the EMT process.It will provide new ideas and potential target sites for the clinical treatment of triple-negative breast cancer with high metastatic potential.