Indoleamine 2,3-dioxygenase 1 Regulates Breast Cancer Tamoxifen Resistance Through IL-6/STAT3

BackgroundAbout 70% of breast cancer patients have positive hormone receptor expression and tamoxifen is the first choice of drugs for endocrine therapy,while 30% of patients develop resistance that leads to treatment failure.IDO1 is a tryptophan metabolism rate-limiting enzyme.At present,studies on the role and mechanism of IDO1 in immune tolerance are relatively sufficient.With the deepening of research,more and more studies show that IDO1 can mediate the occurrence and development of tumors through non-immune tolerance mechanisms.The role of IDO1 in the development and progression of tamoxifen-resistant breast cancer has been rarely reported,and the specific regulatory mechanism has not been clarified.ObjectivesThis study aims to explore the effect of IDO1 on tamoxifen-resistant breast cancer cells,verify whether IDO1 mediates MCF-7-TAMR proliferation and metastasis and promote the occurrence of drug resistance through the IL-6/STAT3 pathway,and explore the molecular mechanism of tamoxifen treatment promoting IDO1 expression.MethodsqRT-PCR and Western blot were used to detect the expression of IDO1 in normal mammary epithelial cells MCF-10 A,breast cancer cells MCF-7-TAMR and tamoxifenresistant breast cancer cells MCF-7-TAMR.Lentivirus transfection technique was used to knock down and overexpress the expression of IDO1 in MCF-7-TAMR.The transfection efficiency was verified by qRT-PCR and Western blot.Cloning and cycle experiments were performed to detect changes in the proliferation ability of MCF-7-TAMR cells after IDO1 knockdown or overexpression,and transwell experiments and scratched experiments were performed to verify changes in the invasion and migration ability of MCF-7-TAMR cells after IDO1 knockdown or overexpression.Western blot showed the expression of cyclicrelated proteins and EMT-related proteins after IDO1 knockdown or overexpression to show the effect of IDO1 on the proliferation and metastasis of MCF-7-TAMR cells.The down-regulated differential expression genes after IDO1 knockdown was obtained by high-throughput sequencing,and the co-expression gene IL-6 was obtained by KEGG analysis and PPI analysis.The secretion of IL-6 in MCF-7-TAMR after IDO1 knockdown or overexpression was detected by ELASA.Western blot was used to detect expression of STAT3,p-STAT3 and ERα.CCK8 was used to evaluate the influence of IDO1 knockdown or overexpression on tamoxifen resistance.The rescue experiment was used to detect whether blocking the IL-6 signaling pathway could counteract the biological behavior in MCF-7-TAMR induced by IDO1 overexpression.Cloning and cycle assay were used to detect the differences between the proliferation status of MCF-7-TAMR cells after up-regulation of IDO1 and inhibition of IL-6 signaling pathway at the same time and overexpression of IDO1 alone.Transwell and scratch assays were used to detect differences in the metastasis and invasion status of MCF-7-TAMR cells after up-regulation of IDO1 and inhibition of IL-6 signaling pathway versus IDO1 overexpression alone.Western blot was used to determine whether inhibition of IL-6signaling pathway could eliminate the changes in the expression of cell cycle,EMT-related proteins and drug-resistant proteins induced by IDO1 overexpression.Subcutaneous xenograft modeling and lung metastasis modeling in nude mice were used to analyze the effect of IDO1 knockdown or IDO1 inhibitor on tamoxifen’s anti-tumor effect.IDO1 inhibition enhanced tamoxifen’s anti-tumor effect and inhibited tumor growth and metastasis.Immunohistochemistry of breast cancer tissue chip was used to verify the expression of IDO1 in Luminal breast cancer.qRT-PCR and Western blot were used to verify the difference of expression of IDO1 in MCF-7 cells before and after tamoxifen treatment.CHIP assay and dual fluorescein assay were used to explore the mechanism of tamoxifen treatment promoting IDO1 up-regulation.qRT-PCR and Western blot were used to detect the regulation of STAT1 on mRNA and protein levels of IDO1.ResultsThis study demonstrated that IDO1 is highly expressed in tamoxifen-resistant breast cancer cells,promoting proliferation and metastasis of resistant cells and mediating tamoxifen resistance through IL-6/STAT3 pathway.This study confirmed that tamoxifen therapy can increase IDO1 expression by promoting IDO1 transcription through STAT1.This provides a new theoretical basis for IDO1 to become a new target for the prevention and treatment of tamoxifen-resistant breast cancer.