Fig. 7: Carmofur in combination with MAPK pathway inhibitor trametinib causes potent TNBC tumor growth inhibition.

A The indicated TNBC cancer cell lines were treated with various concentrations of trametinib for 3 days, and survival was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell survival is presented relative to the survival of DMSO-treated cells. B The indicated TNBC cell lines were treated with indicated concentrations of trametinib and analyzed for their abilities to grow in soft-agar assay. Representative images are shown with a scale bar of 500 µm. C Colony sizes plotted for the experiment shown in (B). D The indicated TNBC cell lines were treated with indicated concentrations of trametinib for 2–4 weeks. Cell survival was measured using a clonogenic assay. Representative images are shown. E TNBC cell lines MDA-MB-231 and MDA-MB-468 were treated with DMSO, 10 µM carmofur alone, 20 nM trametinib alone, or both in combination for 2–4 weeks and analyzed for their abilities to grow in soft-agar assay. Representative images are shown with a scale bar of 500 µm. F Colony sizes plotted for the experiment shown in (E). G MDA-MB-231 cells were subcutaneously injected into the flanks of female NSG mice (n = 3). Mice were treated with vehicle, 20-mg/kg carmofur alone, 2-mg/kg trametinib alone, or both in combination. The average tumor volume was assessed weekly and plotted. H The proposed model for the study—ASAH1 promotes TNBC tumor growth and progression by regulating DUSP5 expression, which inhibits the MAPK pathway. ASAH1 and MAPK pathway can both be targeted via small-molecule inhibitors, either alone or in combination, to provide an effective TNBC therapy. Data represent the mean ± standard error for three biological replicates. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns not significant.