Using Compound Mimic Library To Explore Combination Therapy With Metformin In Triple-negative Breast Cancer

Background and objective:There are restricted effective therapeutic targets for triple-negative breast cancer(TNBC),which lacks expression of estrogen receptor,progesterone receptor and human epidermal growth factor receptor 2.In this way,the prognosis of these patients is usually poor.Exploring new and effective TNBC treatment options is a clinical problem that urgently needs to be solved.Anti-tumour effects of metformin have broad prospects for development.Exploration of a combination treatment plan with metformin and existing targeted drugs may become a breakthrough compared with nowadays treatment of TNBC patients.Currently,CRISPR-Cas9 technology allows large-scale manipulation of genes to screen drug targets and represents an effective method for screening combination drug regimens.Therefore,our study uses CRISPRCas9 library technology to explore the strategy of combination treatment with metformin in TNBC and then explain its mechanism.Our study aims to provide additional treatment options for patients with refractory advanced breast cancer.Methods and results:Our project has three parts.In the first part,our study constructed CRISPR-Cas9 compound mimic library(CCML)with the help of Cambridge anti-cancer compound library and its covered targets.After metformin treatment and library screening,quality control of all samples was good.Then,our study analyzed screening results and found that EGFR,HDAC,and ROCK inhibitors are potential compounds that could combine with metformin.Finally,in vitro experiments found that the combination use of metformin could most strongly induce HDAC inhibitors to apoptosis compared with other inhibitors.In the second part,firstly,our study found that both concentration and time gradient of metformin could induce an increase in apoptosis of SAHA.Besides,metformin and SAHA had a synergistic inhibitory effect on the proliferation of different triple-negative breast cancer cells.Then,our study could not find a synergistic inhibitory effect on treatment of the bottom two potential compounds: ACT-064992,Bosentan and metformin.Finally,in vivo experiments found that the combination of metformin and SAHA can effectively inhibit tumour growth in BALB/c mice.In the third part,through RNA sequencing and real-time fluorescent quantitative,our study found a target named FGFR4.FGFR4 was an insensitive target in treatment of SAHA,while it could turn out to be sensitive when treated plus metformin in triplenegative breast cancer cells.After treatment with concentration or time gradient of SAHA,the FGFR4-JAK-STAT pathway,FGFR4-AKT-ERK and FGFR4-AKT-S6 pathways in different triple-negative breast cancer cells were fully activated,leading to anti-apoptosis of the cells.In addition,our study also found that whether specifically interfering with FGFR4,JAK1,or STAT3 si RNA,the proliferation of triple-negative breast cancer cells was significantly inhibited,and cell apoptosis was also significantly increased;after over expression of FGFR4,the proliferation of triple-negative breast cancer cells was relatively uninhibited,and cell apoptosis was relatively reduced.On the contrary,after treatment with concentration or time gradient of metformin,the FGFR4-AKT-ERK and FGFR4-AKT-S6 pathways in different triple-negative breast cancer cells were inhibited,and apoptosis was induced.In addition,our study also found that after specifically interfering with FGFR4 si RNA,the proliferation of triplenegative breast cancer cells was significantly inhibited,and cell apoptosis was also significantly increased;after overexpression of FGFR4,the proliferation of triplenegative breast cancer cells was relatively not inhibited,apoptosis was also relatively reduced.Besides,through in vitro western blotting experiments and in vivo immunohistochemistry experiments,our study found that the combined use of metformin could down-regulate the activation of FGFR4 and its downstream signalling pathways,which were up-regulated by SAHA alone.To further understand how metformin inhibited the activation of the FGFR signal pathway,through TCGA database information,our study found that,compared with normal breast tissues,FGFR4 and glycolysis-related genes were highly expressed in breast tissue cancer tissues.Besides,compared to the low-expressing FGFR4 group in breast cancer tissues,the high-expressing FGFR4 group also highly expressed glycolysis-related genes.In addition,our study used fluorescent quantitative PCR and glycolysis stress test to detect changes in the m RNA expression of glycolysis-related genes and the extracellular acidification rate.After metformin treatment or specifically interfered with FGFR4siRNA,our study found that the transcription level of glycolysis-related genes was significantly decreased in different triple-negative breast cancer cells,and the rate of extracellular acidification was reduced,indicating that metformin probably inhibited the expression of FGFR4 by inhibiting the glycolysis pathway.Conclusion:With the help of CRISPR-Cas9 library technology,our study found the combination therapy of metformin and the existing targeted drug HDAC inhibitor SAHA.It could produce a synergistic sensitization therapeutic effect.This plan may become a breakthrough compared with nowadays treatment of advanced TNBC patients.In addition,our study pointed out the mechanism of this combination therapy.This treatment plan may be more suitable for TNBC patients with overexpression of FGFR4,and this type of patients is also more sensitive to metabolic inhibitors.Therefore,our study provides a solid theoretical basis for new treatment options for patients with refractory advanced breast cancer.