Elucidating the Regulation and Effectors of the Breast Cancer Oncogene, IKKepsilon

Abstract

The IkappaB kinase epsilon (IKKepsilon, IKKi, IKBKE) is both a regulator of innate immunity and a breast cancer oncogene that is amplified and overexpressed in ~30% of breast cancers. IKKepsilon promotes malignant transformation through the activation of NF-kappaB signaling. In addition, breast cancers that harbor amplifications in the IKBKE gene are dependent on IKKepsilon protein expression for survival. IKKepsilon has been characterized as a non-canonical inhibitor of kappaB kinase (IKK) that activates both the interferon response pathway and NF-kappaB signaling in innate immunity. In this dissertation, I explore both the regulation and effectors of the IKKepsilon kinase in the context of malignant transformation. I found that IKKepsilon is modified and regulated by K63-linked polyubiquitination, a proteasome- and degradation-independent form of ubiquitination, at Lysine 30 and Lysine 401. This modification is essential for IKKepsilon-induced kinase function and IKKepsilon-mediated NF-kappaB activation and malignant transformation. Furthermore, I identified TRAF2 as the K63 ubiquitin E3 ligase that associates with and modifies IKKepsilon. I also found that TBK1, a close family member of IKKepsilon, is also regulated by K63-linked ubiquitination. In collaborative work, we used an unbiased positional scanning peptide library screen to identify two novel downstream targets of IKKepsilon phosphorylation in the context of cancer. Specifically, we found IKKepsilon phosphorylates the tumor suppressor CYLD at Serine 418. CYLD phosphorylation at Ser418 downregulates its deubiquitinase activity and is necessary for IKKepsilon-driven transformation. IKKepsilon also phosphorylates TRAF2 at Serine 11. This activity promotes K63-linked TRAF2 ubiquitination, NF-kappaB activation and is also essential for IKKepsilon-transformation. In addition, breast cancer cells that depend on IKKepsilon expression for survival are also dependent on TRAF2. Together, these observations define an oncogenic network that promotes NF-kappaB-mediated cell transformation through the K63-linked ubiquitination of IKKepsilon and subsequent phosphorylation of two novel substrates, TRAF2 and CYLD.