PKIB Regulates Tamoxifen Resistance Through Inhibition Of Autophagy In Estrogen Receptor-Positive Breast Cancer Cells

Objective: To investigate the molecular targets and specific mechanisms that regulate autophagy and tamoxifen(TAM)resistance in Estrogen Receptor(ER)-positive breast cancer and to provide new ideas for drug development and treatment to reverse TAM resistance.Methods: We used Gene Expression Omnibus(GEO),The Cancer Genome Atlas(TCGA)and other databases and bioinformatics methods to screen the differentially expressed target genes in TAM resistance microarray and TAM sensitivity microarray.The target genes were validated in a TAM-resistant cell line model that was successfully constructed in ER-positive breast cancer cell lines.The knockdown efficiency of the target gene was verified by RTq-PCR and Western Blotting.Autophagy-associated protein expression was detected by Western blotting,transmission electron microscopy was conducted to detect the formation of autophagic vesicles,and CCK8 assay was used to detect the drug sensitivity of PKIB-upregulated cell lines to TAM.The overexpression technique was used to revert the expression of target genes and to verify the target gene overexpression at m RNA and protein levels.And the same method was used to detect changes in autophagy,and CCK8 assay was used to detect changes in sensitivity of overexpressed cell lines to TAM.The bioinformatics website was used to predict the targeting signaling regulatory pathways of PKIB,and the target genes associated with the biological behavior we have validated were selected to validate the mechanism.Collection of tumor tissue samples from ER+ patients before and after TAM treatment for immunohistochemical semi-quantitative experiments.Results: Gene Expression Omnibus(GEO),The Cancer Genome Atlas(TCGA)database and bioinformatics screening indicated that protein kinase inhibitor β(PKIB)from the protein kinase inhibitor(PKI)family may be involved in the regulation of TAM resistance and autophagy in ER-positive breast cancer.(protein kinase inhibitor β,PKIB)may be involved in regulating TAM resistance and autophagy in ER-positive breast cancer.Using si RNA targeting PKIB to knock down the expression level of PKIB in TAM-sensitive cells,Western-blotting detected increased expression of autophagy-associated protein ATG7,increased LC3 I /LC3 Ⅱ ratio,and decreased SQSTM1 expression level;autophagic vesicle formation was observed by electron microscopy,and decreased sensitivity to TAM was detected by the CCK8 assay,which was partially restored by the addition of an autophagy inhibitor(LY294002).Overexpression of PKIB in TAM-resistant cells was detected by Western-blotting,which showed decreased expression of autophagy-related protein ATG7,decreased LC3 Ⅱ/LC3 Ⅰ ratio and increased expression of SQSTM1.Western-blotting experiments showed that the expression of PKIB and its downstream targets,ATG7 and p-CREB,were significantly increased in TAM-resistant cells.In addition,in 20 TAM-resistant patients with recurrent metastases,immunohistochemistry(IHC)results showed that PKIB expression was higher in the primary tumor foci before TAM treatment and significantly lower in TAM-resistant recurrent metastases.CONCLUSION: We demonstrated that in ER-positive breast cancer cells,downregulation of PKIB expression could increase ATG7 transcript levels through the PKA/CREB/ATG7 signaling pathway,thereby enhancing autophagy and ultimately leading to tamoxifen resistance in ER-positive breast cancer cells,and that inhibition of autophagy levels with a combination of autophagy inhibitors could restore TAM drug sensitivity.In conclusion,we propose that PKIB exerts oncogenic effects in ER-positive breast cancer by inhibiting autophagy,and that the combination of autophagy inhibitors can be considered for TAM drug-resistant patients with lower PKIB expression level.