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Cell Syst DOI:10.1016/j.cels.2016.09.001

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers.

Publication TypeJournal Article
Year of Publication2016
AuthorsRosenbluh, J, Mercer, J, Shrestha, Y, Oliver, R, Tamayo, P, Doench, JG, Tirosh, I, Piccioni, F, Hartenian, E, Horn, H, Fagbami, L, Root, DE, Jaffe, J, Lage, K, Boehm, JS, Hahn, WC
JournalCell Syst
Date Published2016 09 28
Keywordsbeta Catenin, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Gene Editing, Genetic Therapy, Humans, Neoplasms, Proteomics, RNA, Guide, RNA, Small Interfering

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.


Alternate JournalCell Syst
PubMed ID27684187
PubMed Central IDPMC5455996
Grant ListU01 CA199253 / CA / NCI NIH HHS / United States
U24 CA194107 / CA / NCI NIH HHS / United States
U01 CA176058 / CA / NCI NIH HHS / United States
R01 CA154480 / CA / NCI NIH HHS / United States
R01 CA121941 / CA / NCI NIH HHS / United States
P50 CA127003 / CA / NCI NIH HHS / United States
R01 MH109903 / MH / NIMH NIH HHS / United States