USP9X-mediated deubiquitination of B-cell CLL/lymphoma 9 potentiates Wnt signaling and promotes breast carcinogenesis.

Hyperactivation of the canonical Wnt-signaling pathway is a prominent feature of a number of human malignancies. Transcriptional activation of this signaling cascade depends on the formation of the β-catenin-B-cell CLL/lymphoma 9 (BCL9)-pygopus (PYGO) family plant homeodomain finger 1 complex, yet how the assembly of this complex is regulated remains to be investigated. Here, using MCF-7, HeLa, HEK293T, MDA-MB-231, and Sf9 cells, along with immunoblotting and immunofluorescence, nano-HPLC-MS/MS, deubiquitination, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assays, we report that BCL9 physically associates with a protein deubiquitinase, ubiquitin-specific peptidase 9, X-linked (USP9X), and that USP9X removes Lys-63-linked polyubiquitin on Lys-212 of BCL9. Importantly, the USP9X-mediated BCL9 deubiquitination facilitated the formation of the β-catenin-BCL9-PYGO complex, thereby potentiating the transcriptional activation of Wnt/β-catenin target genes. We also show that USP9X-mediated BCL9 deubiquitination promotes the proliferation and invasion of breast cancer cells. Together, these results uncover USP9X as a deubiquitinase of BCL9, implicating USP9X in Wnt/β-catenin signaling and breast carcinogenesis.