Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer
Conversion of a death adaptor to a proliferation mediator
Activating mutations in the protein RAS drive cell proliferation and tumor growth. Although best known for mediating cell death signaling through its death domain, when phosphorylated, the adaptor protein FADD promotes cell survival and proliferation. Bowman et al. found that, compared with KRAS mutant mice, KRAS mutant mice engineered to lack FADD or its upstream kinase CK1α developed fewer lung tumors. Lung tissue and cells from KRAS mutant mice had increased abundance of CK1α, phosphorylated FADD, and proliferative markers. In lung tumor samples from patients, expression of FADD was greater in tumors that had mutant KRAS. A CK1α inhibitor prevented FADD from physically interacting with mitotic kinases and suppressed cell proliferation in culture. Thus, blocking the phosphorylation of FADD may be a new strategy for patients with KRAS mutant lung tumors.
Abstract
Genomic amplification of the gene encoding and phosphorylation of the protein FADD (Fas-associated death domain) is associated with poor clinical outcome in lung cancer and in head and neck cancer. Activating mutations in the guanosine triphosphatase RAS promotes cell proliferation in various cancers. Increased abundance of phosphorylated FADD in patient-derived tumor samples predicts poor clinical outcome. Using immunohistochemistry analysis and in vivo imaging of conditional mouse models of KRASG12D-driven lung cancer, we found that the deletion of the gene encoding FADD suppressed tumor growth, reduced the proliferative index of cells, and decreased the activation of downstream effectors of the RAS–MAPK (mitogen-activated protein kinase) pathway that promote the cell cycle, including retinoblastoma (RB) and cyclin D1. In mouse embryonic fibroblasts, the induction of mitosis upon activation of KRAS required FADD and the phosphorylation of FADD by CK1α (casein kinase 1α). Deleting the gene encoding CK1α in KRAS mutant mice abrogated the phosphorylation of FADD and suppressed lung cancer development. Phosphorylated FADD was most abundant during the G2/M phase of the cell cycle, and mass spectrometry revealed that phosphorylated FADD interacted with kinases that mediate the G2/M transition, including PLK1 (Polo-like kinase 1), AURKA (Aurora kinase A), and BUB1 (budding uninhibited by benzimidazoles 1). This interaction was decreased in cells treated with a CKI-7, a CK1α inhibitor. Therefore, as the kinase that phosphorylates FADD downstream of RAS, CK1α may be a therapeutic target for KRAS-driven lung cancer.
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Supplementary Material
Summary
Fig. S1. Fadd-null lesions have lower abundance of phosphorylated ERK1/2 but no detectable difference for an apoptotic marker.
Fig. S2. Fadd-null lesions still express Fadd transgene.
Fig. S3. Increased FADD mRNA correlates with KRAS mutation status in lung cancer patients.
Fig. S4. FADD interacts with proteins involved in the cell cycle.
Fig. S5. Immunohistochemistry of Csnk1a1-null lesions reveals residual CK1α protein.
Data file S1. Mass spectrometry data.
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Copyright © 2015, American Association for the Advancement of Science.
History
Received: 17 June 2014
Accepted: 8 January 2015
Acknowledgments
We thank K. Cho for the Rosa26LSL-Luciferase mice and M. Pasca di Magliano for the KrasLSL-G12D mice. We thank the Lautenberg Center for Immunology at the Hebrew University-Hadassah Medical School and especially Y. Ben-Neriah and E. Pikarsky for their gift of the Csnk1a1fl/fl mice. We are thankful to the Microscopy and Image Analysis Laboratory and FACS cores at the University of Michigan for assisting with all of the microscopy and cell cycle data. Also, thanks to M. Lafferty, J. Stevenson, P. Joseph, K. Vetter, and J. Blossom for their technical assistance. Funding: This work was supported by NIH grants R01CA129623 and P50CA093990. Author contributions: B.M.B., B.D.R., A.R., and S.G. planned and wrote the paper; B.M.B., K.A.S., B.A.H., J.L.B., D.L.D., K.A.H., and S.G. planned and performed experiments; R.M.P. performed pathological analysis of mouse lung cancer histology; C.J.G., D.G.B., and J.Z. provided materials and Fadd−/−; Fadd:GFP mice, expertise, and discussions over the course of the experiments and during the preparation of the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The mass spectrometry data (data file S1) are also available at www.med.umich.edu/cmi/publications_2015.html.
