| Purpose: To explore the effect of novel small molecule tyrosine kinase inhibitor DCC-2036 on triple-negative breast cancer stem cells and related mechanisms to provide potential drug candidates for the treatment of refractory TNBC.Experimental design: 1.MDA-MD-231 and HS-578 T cell lines were cultured and stably passaged;ALDH+ stem/progenitor cell population was detected by flow cytometry,the mammosphere formation experiment,Western Blot detected the expression of stem cell markers.We evaluated the inhibitory effects of DCC-2036 on CSCs of TNBC cell lines MDA-MB-231 and HS-578 T by these experiments.2.Western Blot assay confirmed that the inhibitory effect of DCC-2036 on KLF5 and its downstream target genes in TNBC cells,and explore the effect of KLF5 overexpression on the effect of DCC-2036 on TNBC stem cells by transfecting KLF5 plasmid to determine whether KLF5 mediates DCC-2036 inhibits TNBC stem cells.q RT-PCR,actinomycin(CHX)tracer experiments,and proteasome inhibitor MG132 blocking experiments were used to investigate whether DCC-2036 reduced KLF5 expression by reducing the protein stability of KLF5.Western Blot,q RT-PCR,and other experiments were used to observe the changes in AXL activity and expression and KLF5 after DCC-2036 treatment,and to change the expression of KLF5 and its downstream after knocking down / activating AXL to study whether DCC-2036 reduced KLF5 protein stability by inhibiting AXL.The changes of key proteins of Akt/GSK3β signaling pathway after treatment with DCC-2036 and AXL activating factors were observed by Western Blot experiments.Results: 1.Experiments in vitro cytology demonstrated that DCC-2036 has an inhibitory effect on TNBC stem cells.Flow cytometry results showed that compared with the control group,DCC-2036 significantly reduced the proportion of ALDH+ cell population in TNBC cells MDA-MB-231 and HS-578 T.Mammosphere formation experiments showed that compared with the control group,DCC-2036 also significantly reduced the number and volume of mammosphere formed by both cell lines.Western Blot results showed that DCC-2036 down-regulated the protein expression of stem cell-related markers in MDA-MB-231.2.Mechanistically,we found that DCC-2036 may inhibit TNBC CSCs through the AXL/Akt/GSK-3β/KLF5 axis.(1)Western Blot demonstrated that DCC-2036 significantly inhibited the expression of KLF5 and its downstream target genes Nanog and FGF-BP1 in TNBC cells. Transfection of KLF5 plasmid by Western Blot and mammosphere culture showed that overexpression of KLF5 partially reversed the inhibitory effect of DCC-2036 on TNBC stem cells.(2)we found that DCC-2036 significantly reduced the m RNA level of the KLF5 target gene Nanog,but DCC-2036 did not reduce the KLF5 m RNA level in MDA-MB-231 cells through q RT-PCR technology.Through actinomycin tracer experiments,it was found that compared with the negative control group,DCC-2036 significantly accelerated the degradation of KLF5 in MDA-MB-231 and HS-578 T cells.The proteasome inhibitor MG132 could rescue DCC-2036-induced decrease of KLF5 in two cell lines.(3)Using small interfering RNA(si RNA)to knock down MET or P65 to detect changes in KLF5 in MDA-MB-231 cells,it was found that the expression level of KLF5 protein did not change significantly,while knocking down AXL reduced the KLF5 and its downstream target gene Nanog.At the protein level,MG132 also blocked AXL knockdown-induced down-regulation of KLF5.However,q RT-PCR technology results showed that knocking down AXL significantly reduced the m RNA level of Nanog but did not reduce the m RNA level of KLF5.(4)Western Blot showed that the levels of key proteins in the AXL/Akt/GSK-3β/KLF5 signaling pathway decreased after DCC-2036 treatment,and the AXL/Akt/GSK-3β/KLF5 signaling pathway inhanced expression of protein levels by Gas6(AXL agonist)in MDA-MB-231 and HS-578 T.Conclusion: DCC-2036 inhibits TNBC CSCs by regulating the protein stability of KLF5 by targeting AXL to inhibit Akt/GSK3β signaling pathway. |
PDF Full Text Request