Can losing a protein that restrains growth signaling make cancer cells grow more?

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Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates the degradation of activated EGFR through ubiquitination and also acts as an adaptor, recruiting proteins needed for trafficking.

In this experimental cell-culture and mouse study, the researchers used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with and without epidermal growth factor (EGF) stimulation. They identified over a hundred novel Cbl interactors. A secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) increased EGFR phosphorylation and degradation following EGF stimulation in HeLa cells.

In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling; these effects were reversible with the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking.

FLOT2 interacted with both Cbl and EGFR. The reduction in EGFR levels after FLOT2 loss depended on Cbl: simultaneous knockdown of Cbl and Cbl-b restored EGFR levels. Conversely, FLOT2 overexpression decreased EGFR signaling and growth. In HEK293T cells, overexpression of wildtype (WT) FLOT2 inhibited EGFR phosphorylation following EGF stimulation, whereas overexpression of the soluble G2A FLOT2 mutant did not.

FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. FLOT2 KO increased tumor formation in nude mice and tumor volume in NSG mice, respectively.

Together, the data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, along with subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.