Analysis of epidermal growth factor-induced NF-κB signaling

The nuclear factor kappaB (NF-κB) is a family of transcription factors that control cell survival, cell proliferation, cell differentiation, inflammatory responses, and innate and adaptive immune responses. Its activation is tightly regulated, and incorrect regulation of NF-κB has been linked to a variety of pathological diseases, including cancer initiation and progression. NF-κB is often constitutively activated in cancer cells to promote cell survival, proliferation, migration, and/or epithelial-to-mesenchymal transition (EMT). Although the mechanism of constitutive NF-κB activation in cancer cells is not fully understood, it has been shown that mutation or aberrant expression of epidermal growth factor receptor (EGFR) contributes to this, and the NF-κB activation, in turn, contributes to cell proliferation, survival, metastasis, and drug resistance in various cancers. Recent study from our lab indicates that CARMA3, similar to the function of CARMA1 in mediating antigen receptor-mediated NF-κB activation, plays an essential role in mediating EGFR-induced NF-κB activation. However, the mechanism on how EGFR induces NF-κB activation is not clearly understood. In this chapter, we describe the methods required to test and characterize the role of a potential signaling component in EGFR-induced NF-κB activation.