Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells

Biochim Biophys Acta. 2011 Aug;1813(8):1404-11. doi: 10.1016/j.bbamcr.2011.04.002. Epub 2011 Apr 21.

Abstract

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Apoptosis
  • Base Sequence
  • Caspase 3 / metabolism
  • Cell Cycle
  • Cell Proliferation
  • Colony-Forming Units Assay
  • DNA Primers / genetics
  • Down-Regulation
  • Fusion Proteins, bcr-abl / metabolism
  • Gene Knockdown Techniques
  • Humans
  • Insulin Receptor Substrate Proteins / antagonists & inhibitors*
  • Insulin Receptor Substrate Proteins / genetics
  • Insulin Receptor Substrate Proteins / metabolism
  • K562 Cells
  • MAP Kinase Signaling System
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / metabolism*
  • RNA, Small Interfering / genetics
  • Signal Transduction
  • TOR Serine-Threonine Kinases / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • CRKL protein
  • DNA Primers
  • IRS1 protein, human
  • Insulin Receptor Substrate Proteins
  • Nuclear Proteins
  • RNA, Small Interfering
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Fusion Proteins, bcr-abl
  • Proto-Oncogene Proteins c-akt
  • Casp3 protein, rat
  • Caspase 3