Exploring The Effects And Mechanisms Of Three Small Molecule Inhibitors On The Breast Cancer Cells

Background The treatment of breast cancer is gradually entering the era of precision therapy,and various targeted drugs are emerging.However,chemotherapy is still indispensable to the treatment of some metastatic breast cancers.The treatment of Triple-negative breast cancer(TNBC)has always been one of the most challenging aspects of breast cancer.The lack of hormone receptors and HER2 expression leading to a chronic lack of effective treatments for TNBC other than chemotherapy.LIM kinase 1(LIMK1)and p21-activated kinase 4(PAK4)are often over-expressed in breast tumors,which causes aggressive cancer phenotypes and unfavorable clinical outcomes.In addition to the well-defined role in regulating cell division,proliferation and invasion,the two kinases promote activation of the MAPK pathway and cause endocrine resistance through phosphorylating estrogen receptor alpha(ER?).PAK4specifically phosphorylates LIMK1 and its functional partners,indicating possible value of suppressing both kinases in cancers that over-express PAK4 and/or LIMK1.Ubiquitin-specific protease 47(USP47),which is overexpressed in many kinds of cancers,promotes epithelial mesenchymal transition(EMT),regulates DNA repair,and maintains genomic stability in tumor cells,and is associated with poor prognosis.Analysis of publicly available databases shows that USP47 is overexpressed in breast cancer and predicts a poorer prognosis.Methods Firstly,our study investigated whether there were synergistic effects of LIMKi 3 and PF-3758309 in breast cancer cell lines with different molecular type by cell viability assay.Then cell cycle was tested using single drug or combination against Luminal type cell lines.Western blot was used to detect the effect of drugs on kinase phosphorylation,ER?phosphorylation and changes in C-MYC expression.The BT474cell line was used to construct xenograft tumor models.Single or combination treatments were applied to investigate whether the drugs have the same synergistic effect in vivo.Indicators affecting the drug sensitivity of LIMKi 3 and PF-3758309were screened using bioinformatic analysis,and the z GARP score combined with the drug IC50 was used to determine this.Secondly,USP47 inhibitor P22077 was used on triple-negative breast cancer cell lines.Cell viability assay,migration,cell cycle and apoptosis were tested to prove the effects of P22077 in vitro.USP47 knockdown transplant tumors and Py MT^+/-/USP47^-/-knockout mouse were used to investigate whether knockdown of USP47 could inhibit breast cancer development in vivo.Results Firstly,we assessed the impact of combining LIMK1 inhibitor LIMKi 3 and PAK4 inhibitor PF-3758309 in preclinical breast cancer models.LIMK1 and PAK4pharmacological inhibition synergistically reduced the survival of various cancer cell lines,exhibiting specific efficacy in luminal and HER2-enriched models.The combination of the two drugs better inhibited the activation of ER?phosphorylation and C-MYC expression,and suppressed development and ER?-driven signals in a BT474 xenograft model.In silico analysis demonstrated the cell lines with reliance on LIMK1 were the most prone to be susceptible to PAK4 inhibition.Secondly,we proved that P22077 is able to inhibit the proliferation and migration of TNBC cells and suppress the expression of USP47 protein in vitro.Furthermore,P22077 induced cell cycle arrest and apoptosis.Knockdown of USP47 inhibited TNBC cell proliferation and migration in vitro.In addition,knockdown of USP47 significantly prolonged survival and affected tumor size in vivo.Conclusion Double LIMK1 and PAK4 targeting therapy can be a successful therapeutic strategy for breast cancer,with a unique efficiency in the subtypes of luminal and HER2-enriched tumors.Targeting USP47 may become a new targeted therapeutic for TNBC and provide strategies for the treatment of TNBC.