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

Cell Syst. 2016 Sep 28;3(3):302-316.e4. doi: 10.1016/j.cels.2016.09.001.

Abstract

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.

Keywords: CRISPR-Cas9; RNAi; genetic interactions; protein-protein interactions; β-catenin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing
  • Genetic Therapy
  • Humans
  • Neoplasms
  • Proteomics*
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Small Interfering
  • beta Catenin

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Small Interfering
  • beta Catenin