A genome-wide RNAi screen for polypeptides that alter rpS6 phosphorylation

Methods Mol Biol. 2012:821:187-214. doi: 10.1007/978-1-61779-430-8_11.

Abstract

Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have attempted to identify the cellular components necessary to maintain the activity of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, using a whole genome approach involving RNAi-induced depletion of cellular polypeptides. We have used a pancreatic ductal adenocarcinoma (PDAC) cell line, Mia-PaCa for this screen; as with many pancreatic cancers, these cells exhibit constitutive activation of mTORC1. PDAC is the most common form of pancreatic cancer and the 5-year survival rate remains 3-5% despite current nonspecific and targeted therapies. Although rapamycin-related mTOR inhibitors have yet to demonstrate encouraging clinical responses, it is now evident that this class of compounds is capable of only partial mTORC1 inhibition. Identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new targets and lead to the development of beneficial therapies for pancreatic cancer.

Publication types

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

MeSH terms

  • Adenocarcinoma
  • Carcinoma, Pancreatic Ductal / enzymology*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Fluorescent Antibody Technique / methods*
  • Genome, Human
  • Humans
  • Mechanistic Target of Rapamycin Complex 1
  • Multiprotein Complexes
  • Pancreatic Neoplasms / enzymology*
  • Phosphorylation
  • Proteins / antagonists & inhibitors
  • Proteins / genetics
  • Proteins / metabolism*
  • RNA Interference*
  • Ribosomal Protein S6 / metabolism
  • Signal Transduction
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases
  • Transfection / methods*

Substances

  • Multiprotein Complexes
  • Proteins
  • Ribosomal Protein S6
  • Mechanistic Target of Rapamycin Complex 1
  • TOR Serine-Threonine Kinases
  • Sirolimus