X-linked inhibitor of apoptosis protein and its E3 ligase activity promote transforming growth factor-β-mediated nuclear factor-κB activation during breast cancer progression

Abstract

The precise sequence of events that enable mammary tumori- genesis to convert transforming growth factor-β (TGF-β) from a tumor suppressor to a tumor promoter remains incompletely understood. We show here that X-linked inhibitor of apoptosis protem (xIAP) is essential for the ability of TGF-β to stimulate nuclear factor-κB (NF-κB) in metastatic 4T1 breast cancer cells. Indeed whereas TGF-β suppressed NF-κB activity in normal mammary epithelial cells, those engineered to overexpress xIAP demonstrated activation of NF-κB when stimulated with TGF-β. Additionally up-regulated xIAP expression also potentiated the basal and TGF-β-stimulated transcriptional activities of Smad2/3 and NF-κB Mechanistically xIAP (i) interacted physically with the TGF-β type I receptor, (ii) mediated the ubiquitination of TGF-β- activated kinase 1 (TAK1), and (iii) facilitated the formation of complexes between TAK1-binding protein 1 (TAB1) and IκB kinase β that enabled TGF-β to activate p65/RelA and to induce the expression of prometastatic (i.e. cyclooxygenase-2 and plasminogen activator inhibitor-1) and prosurvival (i.e. survivin) genes. We further observed that inhibiting the E3 ubiquitin ligase function of xIAP or expressing a mutant ubiquitin protein (i.e. K63R-ubiquitin) was capable of blocking xIAP- and TGF-β-mediated activation of NF-κB. Functionally xIAP deficiency dramatically reduced the coupling of TGF-β to Smad2/3 in NMuMG cells as well as inhibited their expression of mesenchymal markers in response to TGF-β. More importantly, xIAP deficiency also abrogated the formation of TAB1-IκB kinase β complexes in 4T1 breast cancer cells, thereby diminishing their activation of NF-κB, their expression of prosurvival/metastatic genes, their invasion through synthetic basement membranes, and their growth in soft agar. Collectively our findings have defined a novel role for xIAP in mediating oncogenic signaling by TGF-β in breast cancer cells. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.