SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors.

Abstract

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.

• Authors: Lee Roth, Swati Srivastava, Moshit Lindzen, Aldema Sas-Chen, Michal Sheffer, Mattia Lauriola, Yehoshua Enuka, Ashish Noronha, Maicol Mancini, Sara Lavi, Gabi Tarcic, Gur Pines, Nava Nevo, Ori Heyman, Tamar Ziv, Oscar M Rueda, Davide Gnocchi, Eli Pikarsky, Arie Admon, Carlos Caldas, Yosef Yarden
• Journal: Science signaling (Sci Signal) Vol. 11 Issue 515 (01 30 2018) ISSN: 1937-9145 [Electronic] United States
• PMID: 29382783 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Chemical References

• Autoantigens
• Filamins
• Non-Fibrillar Collagens
• Epidermal Growth Factor
• EGFR protein, human
• ErbB Receptors

Topics

• Actin Cytoskeleton (metabolism)
• Animals
• Autoantigens (genetics, metabolism)
• Breast (drug effects, metabolism, pathology)
• Breast Neoplasms (drug therapy, metabolism, pathology)
• Cell Movement
• Cell Proliferation
• Cells, Cultured
• Epidermal Growth Factor (pharmacology)
• ErbB Receptors (metabolism)
• Female
• Filamins (genetics, metabolism)
• Humans
• Isotope Labeling
• Mice
• Mice, Nude
• Non-Fibrillar Collagens (genetics, metabolism)
• Phosphorylation
• Protein Binding
• Proteomics (methods)
• Xenograft Model Antitumor Assays
• Collagen Type XVII