Abstract 90: OTUD7B activates the caspase 8-RIP1-NEMO complex-regulated NF-kB pathway to promote triple-negative breast cancer metastasis

Abstract Background: Triple negative breast cancer (TNBC) is a highly metastatic BC subtype and refers to a poorer prognosis of BCs. Distant metastasis of TNBCs to other organs, e.g. lungs and brain, risks the patient’s life. However, the molecular mechanism underlying TNBC metastatic progression is still under investigation. The aim of this study was made to delineate the role of ovarian tumor deubiquitinating enzyme 7B (OTUD7B) in the molecular mechanism for TNBC metastasis. Methods: Site-directed mutagenesis was performed to confirm that the deubiquitinating activity of OTUD7B is required for promoting TNBC metastatic potentials. Human Ubiquitin Array kit was employed to identify the potential target of OTUD7B in the highly metastatic TNBC cell line MDA-MB231. Pathway enrichment analysis was utilized to predict the possible pathways involved in TNBC metastasis. Pulldown assay was utilized to validate the deubiquitinating activity of OTUD7B towards the identified protein targets. Cycloheximide-chased assay was used to measure the degradation of identified protein targets in response to OTUD7B knockdown in MDA-MB231 cells and its overexpression in poorly metastatic TNBC cell line HCC1937. Results: Site-directed mutagenesis indicated that the mutation of Asn193 to Leu or Met, which leads to the loss of OTUD7B deubiquitinating activity, as compared to wild-type gene fails to promote cellular migration ability and lung colony-forming capacity of the tested TNBC cells. The employment of Human Ubiquitin Array kit demonstrated that Caspase-8 and NEMO, also named IKKγ, are the potential targets for OTUD7B deubiquitinating activity in MDA-MB231 cells. Pathway enrichment analysis predicted the pathway in which NF-κB activation through FADD/RIP-1 axis mediated by Caspase 8 is probably activated in the primary tumor from TNBC patients with lymph node metastasis. Pulldown assay further confirmed the deubiquitinating activity of OTUD7B towards Caspase-8 and NEMO in TNBC cells. Cycloheximide-chased assay revealed that OTUD7B knockdown accelerates protein degradation of Caspase-8 and NEMO in MDA-MB231 cells. The pharmaceutical inhibition of NEMO complex with IKKα/β dramatically suppressed NF-κB activity and metastatic potentials in the OTUD7B-overexpressing HCC1937 cells. Importantly the signature of combining lower levels of cleaved/active Caspase-8 and higher levels of pS536-NF-κB was found to highly correlate with lymph node metastasis and a poorer overall survival rate in TNBC patients. Conclusion: Our results suggest that OTUD7B upregulation promotes TNBC metastasis by recruiting the Caspase-8-RIP1-NEMO axis-activated NF-κB pathway. Citation Format: Hui-Yu Lin, Kun-Ju Chen, Yuan-Feng Lin. OTUD7B activates the caspase 8-RIP1-NEMO complex-regulated NF-kB pathway to promote triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 90.