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in order to investigate the potential role of ripk1 in tnbc sensitivity to smac mimetics, we knocked down ripk1 using rnai in four different tnbc cell lines (mda-mb-231, bt549, mda-mb-468, and hcc1806). we found that downregulation of ripk1 had no effect on the viability of tnbc cells alone, but markedly enhanced the killing effect of smac mimetics in all tnbc cells. we further tested whether downregulation of ripk1 could enhance smac mimetic-induced apoptosis by measuring the cleavage of caspase-8, caspase-3 and parp in all four tnbc cell lines. knockdown of ripk1 significantly enhanced smac mimetic-induced cleavage of caspase-8, caspase-3 and parp (fig. 4d ). these results suggest that downregulation of ripk1 enhances the killing effect of smac mimetics in tnbc cells through enhanced caspase-dependent apoptosis.
nsa is a covalent inhibitor of ripk1 that covalently binds to the ripk1 kinase domain [ 27 ]. nsa treatment of mda-mb-231, bt549, mda-mb-468, and hcc1806 cells had no effect on cell viability or smac mimetic-induced apoptosis. however, treatment with nsa significantly enhanced the killing effect of smac mimetics on all tnbc cells in a dose-dependent manner (fig. 5a ). we also observed that nsa significantly enhanced the killing effect of smac mimetics in mda-mb-231, bt549, mda-mb-468 and hcc1806 cells when combined with gsk 782 (fig. 5b ). our results suggest that nsa enhances smac mimetic-induced apoptosis in tnbc cells by inhibiting ripk1.
to further assess the generality of our finding, we tested the effects of nsa on other tnbc cell lines including mda-mb-231, bt549, mda-mb-468 and hcc1937. our results showed that nsa enhanced smac mimetic-induced apoptosis in these cell lines (fig. 5c and supplementary fig. 4).
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