CYLD调控凝血酶诱导的p38-p65信号传导以抑制乳腺癌进展

G-protein coupled receptors (GPCRs) are a large and diverse family of cell surface receptors that regulate various physiological responses. Dysregulation of GPCR signaling is associated with multiple diseases, making this receptor family the largest target class of FDA-approved drugs. However, the mechanisms that regulate GPCR signaling are not well defined, and thus important to understand for improving the development of GPCR-targeted drugs. While phosphorylation is recognized as a key mechanism in GPCR regulation, GPCRs are also subject to ubiquitination, which is best known to promote lysosomal degradation. However, we showed that protease-activated receptor 1 (PAR1), which, upon thrombin stimulation undergoes ubiquitination and promotes non-canonical TAB1/TAB2 dependent activation of the p38 MAPK pathway on endosomes. This paradigm underscores a critical limitation of current therapeutic strategies, which predominantly target plasma membrane-initiated signaling while overlooking the contributions of endosomal signaling pathways. Although several E3 ligases have been identified to ubiquitinate GPCRs, the deubiquitinases that reverse this modification remain poorly defined, leaving a major gap in our ability to modulate GPCR signaling with precision in inflammatory diseases. Thrombin-activated PAR1 coupling to endosomal p38 signaling axis provides a powerful model to study how ubiquitination and deubiquitination regulate GPCR signaling. We identified the deubiquitinase CYLD as a key regulator of thrombin-induced PAR1-p38 signaling in endothelial and HeLa cells. Extending these findings to cancer, I demonstrated that thrombin activates p38 in multiple triple-negative breast cancer (TNBC) cell lines where PAR1 is highly expressed. In TNBC cells, CYLD knockdown increased both basal/thrombin-induced phosphorylation of p38 MAPK and p65. Using siRNA and pharmacological inhibitors, I further demonstrated that thrombin-induced p38 signaling functions upstream of NF-κB/p65, with co-immunoprecipitation confirming that p38 directly co-associates and phosphorylates p65 rather than other NF-κB pathway components. Downstream, this pathway drives nuclear translocation of phosphorylated p65, revealed by immunofluorescence and shown to be p38-dependent, as nuclear localization was abrogated by the p38 inhibitor BIRB 796. Functionally, thrombin-p38-p65 signaling upregulated pro-inflammatory cytokines including IL-6, IL-8, IL-1alpha, and IL-1beta, as detected by human cytokine arrays and validated by RT-PCR. Finally, CYLD knockdown enhanced TNBC cell proliferation and migration, demonstrating that CYLD suppresses TNBC growth by inhibiting thrombin-p38-p65-signaling. Collectively, these findings reveal a novel mechanism by which CYLD inhibits TNBC progression through regulation of thrombin-p38-p65 signaling.