Study On The Mechanism Of PHB Regulating The Sensitivity Of Triple Negative Breast Cancer To Docetaxel Through STAT3/cGAS-STING Pathway

Background The incidence of breast cancer ranks the first among female malignant tumors and has become the most common malignancies in the world.Triple negative breast cancer(TNBC)is the most aggressive and highly heterogeneous subtype of breast cancer,accounting for 15%-20%,but resulting in the highest mortality rate of all subtypes.Taxanes,anthracyclines and platinum-based chemotherapy is the main or even the only choice for the systemic treatment of TNBC.Docetaxel(DTX),a representative of the second generation of taxanes,is widely used in the adjuvant and neoadjuvant chemotherapy of breast cancer mainly by inhibiting the proliferation of tumor cells.However,most patients with advanced TNBC develop reduced sensitivity to DTX therapy or even resistance.Prohibitin(PHB)is a member of the membrane protein family widely distributed in a variety of cells.It has been confirmed to be related to the occurrence and development of a variety of malignant tumors and treatment resistance,but its effect on TNBC and DTX treatment of TNBC is still unknown.ObjectiveThis research aimed to explore the expression of PHB in TNBC and its relationship with prognosis,and study the effect and potential mechanism of PHB on the sensitivity of DTX treatment in TNBC.Materials and Methods(1)The Cancer Genome Atlas(TCGA)database was used to explore the expression of PHB in TNBC and its relationship with clinicopathological characteristics of patients.Western Blot(WB)and immunohistochemistry were used to validate the expression of PHB.TNBC cell lines,MDA-MB-231 and MDA-MB-468 were transfected with si RNA to knock down the expression of PHB and establish stable expression cell lines.The effects on cell proliferation,migration,invasion,apoptosis and subcutaneous xenograft model in nude mice were verified.(2)The expression of PHB in DTX-resistant TNBC tissues was verified by immunohistochemistry.TNBC cell lines,MDA-MB-231 and MDA-MB-468,with stable PHB overexpression and low expression were constructed and then treated with DTX,respectively.The changes of cell proliferation,migration,invasion,apoptosis and subcutaneous xenograft model in nude mice were observed to explore the effect of PHB on the sensitivity of DTX treatment in TNBC.(3)Cell transcriptome sequencing,q RT-PCR and WB were used to detect the expression of genes and proteins in potential related pathways,and to explore the potential mechanism of PHB affecting the biological behavior of TNBC and changing the sensitivity of DTX treatment.Results(1)Bioinformatics analysis based on TCGA showed that PHB was highly expressed in TNBC,which was associated with various clinicopathological features such as clinical pathological stage and molecular subtype.Overexpression of PHB could lead to worse recurrence-free survival(RFS)and distant metastasis-free survival(DMFS).WB detection of tissue and cellular proteins verified the high expression of PHB in TNBC.Phenotypic experiments confirmed that knocking down PHB could inhibit the proliferation,migration,invasion and subcutaneous tumorigenesis of MDA-MB-231 and MDA-MB-468,and promoted cell apoptosis.(2)PHB expression is significantly higher in DTX-resistant TNBC group.Knocking down PHB enhanced the inhibitory effect of DTX on the cell proliferation,migration,invasion,subcutaneous tumor formation in nude mice and the promotion of cell apoptosis.Overexpression of PHB reduced the inhibitory effect of DTX on the proliferation,migration,invasion,subcutaneous tumor formation in nude mice and the promotion of cell apoptosis.(3)Transcriptome sequencing confirmed that downregulation of PHB was significantly associated with enrichment of c GAS-STING and interferon signaling pathways.And down-regulation of PHB significantly inhibited the activation of STAT3.Co-Immunoprecipitation(Co-IP)confirmed the direct interaction between PHB and STAT3.Overexpression of STAT3 weaken the effect of PHB downregulation on cell phenotype and tumorigenesis ability,and blocked the expression changes of related pathway molecules caused by PHB downregulation.And downregulation of PHB and or STAT3 could induce the activation of downstream molecules and the expression of downstream related genes of c GAS-STING,while further knockdown of c GAS or STING could block the activation of downstream molecules.ConclusionThis study confirmed that high expression of PHB in TNBC leads to a worse prognosis.Targeted knockdown of PHB can enhance the sensitivity of TNBC to DTX treatment through STAT3/c GAS-STING related pathway.