FAM83B mediates EGFR- and RAS-driven oncogenic transformation
Rocky Cipriano, … , George R. Stark, Mark W. Jackson
Rocky Cipriano, … , George R. Stark, Mark W. Jackson
Published August 13, 2012
Citation Information: J Clin Invest. 2012;122(9):3197-3210. https://doi.org/10.1172/JCI60517.
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Research Article

FAM83B mediates EGFR- and RAS-driven oncogenic transformation

Abstract

Aberrant regulation of growth signaling is a hallmark of cancer development that often occurs through the constitutive activation of growth factor receptors or their downstream effectors. Using validation-based insertional mutagenesis (VBIM), we identified family with sequence similarity 83, member B (FAM83B), based on its ability to substitute for RAS in the transformation of immortalized human mammary epithelial cells (HMECs). We found that FAM83B coprecipitated with a downstream effector of RAS, CRAF. Binding of FAM83B with CRAF disrupted CRAF/14-3-3 interactions and increased CRAF membrane localization, resulting in elevated MAPK and mammalian target of rapamycin (mTOR) signaling. Ablation of FAM83B inhibited the proliferation and malignant phenotype of tumor-derived cells or RAS-transformed HMECs, implicating FAM83B as a key intermediary in EGFR/RAS/MAPK signaling. Analysis of human tumor specimens revealed that FAM83B expression was significantly elevated in cancer and was associated with specific cancer subtypes, increased tumor grade, and decreased overall survival. Cumulatively, these results suggest that FAM83B is an oncogene and potentially represents a new target for therapeutic intervention.

Authors

Rocky Cipriano, James Graham, Kristy L.S. Miskimen, Benjamin L. Bryson, Ronald C. Bruntz, Sarah A. Scott, H. Alex Brown, George R. Stark, Mark W. Jackson

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Figure 5

FAM83B and transformation.

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FAM83B and transformation. (A) Schematic of the FAM83B protein, denoting...
(A) Schematic of the FAM83B protein, denoting the domain of unknown function 1669 (DUF1669), the putative PLD-like motif compared with a consensus PLD motif, and the deletion mutants analyzed here. Summary of the FAM83B constructs that drive HMEC AIG. (B and C) The DUF1669 drives AIG. HME1 cells expressing full-length (FL; also denoted WT) FAM83B or FAM83B deletion mutants encoding amino acids 1–482 and 1–284 (denoted 482 and 284, respectively) were assessed for AIG. (D) Immunoblot analysis of FLAG and GAPDH in HME1 cells expressing vector, FAM83B-284, FAM83B-482, the deletion of the DUF1669 (1–283 amino acids), and full-length FAM83B. (E) FAM83B lacks conventional PLD activity. HEK293-TREx cells were transfected with an expression plasmid encoding myc-tagged FAM83B or vector 48 hours prior to cell lysis and immunoprecipitation with a myc-specific antibody. Protein-G agarose-bound myc-FAM83B was assayed for phosphatidylcholine hydrolysis in the presence of known PLD1 activators ARF GTPase and PKC. Myc immunoprecipitates from vector or myc-PLD2–transfected cells served as negative and positive controls, respectively. The graph is a representative figure from the HEK293 immunoprecipitations (n = 3 experiments). (F) HME1 cells expressing GFP or FAM83B were grown as 3D cultures in lrBM. Images were taken, and colony size was determined using MetaMorph image quantitation software. Original magnification, ×10. (G) FAM83B expression activated MAPK and mTOR signaling. Immunoblot analysis of phospho-ERK1/2, phospho-S6K, phospho–4E-BP1, ERK1/2, and GAPDH in HME1 cells expressing GFP or FAM83B. (H) Immunoblot analysis of phospho-ERK1/2, ERK1/2, and GAPDH in HME1 cells expressing GFP, full-length FAM83B, or FAM83B–Δ-DUF.