FADD and its Phosphorylation Mediate Mitogenic Signaling in Mutant Kras Tumors.

Abstract

Fas-Associated Death Domain (FADD) through its function as an adapter protein is a key mediator of cell death. Paradoxically, amplification of its locus on 11q13.3, increased mRNA and protein expression, phosphorylation at Serine 194 (S194) and subsequent nuclear translocation has been linked with aggressive disease and poor outcomes in patients with lung as well as head and neck as cancer. To investigate a role for FADD in oncogenesis, we genetically engineered a conditional mouse model of mutant Kras-driven lung cancer wherein Fadd expression could be abrogated simultaneously with activation of mutant Kras. In the absence of FADD, a decrease in the number of lung lesions and a slower growth rate was observed. Tumors from Fadd-null animals exhibited a lower proliferative index and muted activation of downstream effectors of the RAS-mitogen-activated protein kinase (MAPK) pathway, including phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2), phosphorylated retinoblastoma (pRB) and Cyclin D1, indicating alterations in cell proliferation. In support, analysis of the FADD-interactome revealed its association with Polo-like Kinase 1(PLK1), Aurora Kinase A (AURKA), Budding Uninhibited by Benzimidazoles 1(BUB1) and CDC20, important regulators of the G2/M transition. A requirement for FADD in G2/M was further supported by the arrest of Fadd-null mouse embryonic fibroblasts at G2/M and the observation that phosphorylation of FADD at S194 preceded phosphorylation of phospho-histone H3. Heterologous expression of FADD in Fadd-null cells restored the progression of cells through G2/M and proliferation of mutant Kras expressing cells. Conditional deletion of Casein Kinase 1 alpha simultaneous with activation of mutant Kras, identified it as the kinase required for FADD-phosphorylation. Mice lacking Casein Kinase 1 alpha failed to develop adenomas but had hyperplastic lesions despite the expression of mutant Kras. These studies identify the significance of FADD and its phosphorylation by Casein Kinase 1 alpha in Kras-mediated oncogenic transformation and therefore a target for therapeutic intervention in lung cancer.