ABSTRACT
EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.
Subject(s)
CRISPR-Cas Systems , Carcinoma, Non-Small-Cell Lung/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , A549 Cells , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line, Tumor , Cullin Proteins , ErbB Receptors/genetics , Female , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Guanine Nucleotide Exchange Factors/genetics , HEK293 Cells , Humans , Methionyl Aminopeptidases/metabolism , Mice , Mice, Nude , Receptors, Lysophosphatidic Acid/metabolism , Signal Transduction , Transcription Factors/metabolism , Transcriptome , Ubiquitin-Protein Ligases/genetics , YAP-Signaling Proteins , rhoA GTP-Binding Protein/metabolismABSTRACT
We have used a combination of SDS-PAGE and LTQ-Orbitrap MS to explore the proteome of the highly invasive MDA-MB-231 breast cancer cell line. Based on about 520 000 MS/MS spectra, a total of 3481 proteins were identified and subsequently classified according to their cellular distribution and molecular function. Interestingly, a large proportion of proteins (38%) were from cellular membranes and we were able to characterize numerous proteins involved in cancer initiation and progression such as the tumor suppressor p53 and the epidermal growth factor receptor. Together, this study represents the largest proteome database of breast cancer cells realized to date and demonstrates the value of using Orbitrap MS for deeper proteome analysis.
