The deubiquitinase CYLD inhibits thrombin-induced p38 MAPK-p65 NF-κB signaling and inflammatory cytokine production to suppress triple-negative breast cancer progression.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by chronic inflammation with limited therapeutic options. Although thrombin activation of protease-activated receptor-1 (PAR1) promotes ubiquitin-dependent signaling and inflammatory responses in endothelial cells, its contribution to inflammatory effects in TNBC remain poorly understood. Here, we show that loss of the deubiquitinase cylindromatosis (CYLD) markedly increases basal phosphorylation of p38 mitogen-activated protein kinase and p65 nuclear factor kappa B subunit to levels comparable to thrombin stimulation, without further enhancement by thrombin, indicating that CYLD primarily restrains constitutive inflammatory activity. We also show that thrombin-activation of PAR1 promotes p38 signaling through an autophosphorylation-dependent mechanism in TNBC, leading to p65 nuclear factor kappa B subunit phosphorylation and nuclear accumulation independent of canonical inhibitor of κBα degradation. This non-canonical signaling drives expression of pro-inflammatory interleukin (IL) cytokines including IL-6, IL-8, IL-1α, and IL-1β. Functionally, IL-6 and IL-8 contribute to thrombin-induced TNBC proliferation and migration, respectively. Together, these findings define a novel CYLD-regulated PAR1-p38-p65 signaling axis that promotes inflammatory cytokine production and tumor-associated phenotypes such as cell proliferation and migration, identifying multiple druggable nodes for therapeutic targeting in aggressive breast cancers.