A multistep high-content screening approach to identify novel functionally relevant target genes in pancreatic cancer

PLoS One. 2015 Apr 7;10(4):e0122946. doi: 10.1371/journal.pone.0122946. eCollection 2015.

Abstract

In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of 'reverse transfection array' technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from "druggable" gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Cell Line, Tumor
  • Cell Proliferation
  • Fetus
  • G-Protein-Coupled Receptor Kinase 2 / genetics
  • G-Protein-Coupled Receptor Kinase 2 / metabolism
  • Gene Expression Profiling / methods*
  • Humans
  • Intracellular Space / metabolism
  • Pancreatic Neoplasms / genetics*
  • Pancreatic Neoplasms / pathology
  • Protein Transport
  • Proto-Oncogene Proteins c-crk / genetics
  • Proto-Oncogene Proteins c-crk / metabolism
  • Reverse Transcription

Substances

  • Proto-Oncogene Proteins c-crk
  • GRK2 protein, human
  • G-Protein-Coupled Receptor Kinase 2

Grants and funding

This work was funded in part by EU FP6 grant LSHB-CT-2006-018771 (Integrated Project ”MolDiag-Paca”), the German federal ministry of education and research (BMBF) within the framework of the program of medical genome research (PaCa-Net; project ID PKB-01GS08 and 01GS08178), the German Research Foundation (DFG; grant Bu 1536/3-1 to M.B.), and EU FP7 grant no. 602783 (large-scale integrated project “CAM-PaC”). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.