LAUSR

EGF receptor (EGFR) signaling is dysregulated by gene amplification or activating mutations of the EGFR in a variety of human malignancies. Activation of the EGFR by EGF leads to recruitment of the ubiquitin ligase Cbl, which ubiquitinates EGFR targeting it for degradation and acts as an adaptor, recruiting additional proteins required for EGFR internalization and trafficking to the lysosome. However, the detailed molecular mechanisms remain elusive. Complete characterization of proteins involved in EGFR trafficking and degradation may uncover additional mechanisms of EGFR dysregulation in cancer. To characterize the EGFR degradative pathway, we investigated protein complexes formed on Cbl with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry. By labeling proteins with stable isotopes in culture, we compared complexes in 3 groups: 1) complexes formed on Cbl without EGF stimulation (control), 2) complexes formed on Cbl with EGF stimulation for 30 min and 3) complexes formed on Cbl with EGF stimulation in the presence of YM201636, a trafficking inhibitor, which was added to prevent degradation of the Cbl/EGFR complex and to enrich it for other proteins important for EGFR trafficking. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed significantly with the treatments. Fifty of these proteins were selected for RNAi knockdown to examine their effects on EGFR degradation. We found that knockdown of Flotillin 2 (FLOT2) protein, a major scaffold protein within caveolar lipid rafts, led to increased phosphorylation and accelerated degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. Conversely, overexpression of FLOT2 in HEK293T inhibited EGFR phosphorylation and formation of the EGFR/Cbl complex upon EGF stimulation and led to decreased EGFR ubiquitination and degradation. CRISPR knockout of FLOT2 in HeLa cells led to decreased steady-state levels of EGFR, increased phosphorylation of EGFR and ERK1/2 compared to non-targeting CRISPR control. Stable overexpression of FLOT2 in HeLa cells increased steady-state levels of EGFR, but decreased EGFR phosphorylation and ubiquitination upon EGF stimulation. FLOT2 is frequently amplified and overexpressed in breast, endometrial and lung cancers. In a lung adenocarcinoma cell line H441, in which FLOT2 gene is amplified, knockdown of FLOT2 increased EGFR phosphorylation and ubiquitination, as well as EGFR signaling, as detected by the phosphorylation of ERK1/2. Our data indicate that FLOT2 negatively regulates EGFR phosphorylation, ubiquitination and degradation. Although it is not clear how FLOT2 amplification could contribute to cancer pathogenesis, we speculate that studying how FLOT2 regulates EGFR signaling could lead to a better understanding of its role in normal physiology and cancer. Citation Format: Mariya Liyasova, Xu Zhang, Udayan Guha, Stanley Lipkowitz. Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2506.