IGFBP-2 nuclear translocation is mediated by a functional NLS sequence and is essential for its pro-tumorigenic actions in cancer cells

Oncogene. 2014 Jan 30;33(5):578-88. doi: 10.1038/onc.2012.630. Epub 2013 Feb 25.

Abstract

IGFBP-2 is highly expressed in both the serum and tumor tissues of most cancers, and is considered one of the most significant genes in the signature of major cancers. IGFBP-2 mainly modulates IGF actions in the pericellular space; however, there is considerable evidence to suggest that IGFBP-2 may also act independently of the IGFs. These IGF-independent actions of IGFBP-2 are exerted either via interactions at the cell surface or intracellularly, via interaction with cytoplasmic or nuclear-binding partners. The precise mechanism underlying the intracellular/intranuclear localization of IGFBP-2 remains unclear. In this study, we investigated IGFBP-2 nuclear localization in several common cancer cells with the aim of dissecting the mechanism of its nuclear trafficking. IGFBP-2 is detected in the nuclei of common cancer cells, including breast, prostate and several neuroblastoma cell lines, using cell fractionation and confocal microscopy. Via nuclear import assays, we show that nuclear entry of IGFBP-2 is mediated by the classical nuclear import mechanisms, primarily through importin-α, as demonstrated by the use of blocking, competition and co-immunoprecipitation assays. Bioinformatics analysis of the IGFBP-2 protein sequence with PSORT II identified a classical nuclear localization signal (cNLS) sequence at 179PKKLRPP185, within the IGFBP-2 linker domain, mutagenesis of which abolishes IGFBP-2 nuclear import. Accordingly, the NLSmutIGFBP-2 fails to activate the VEGF promoter, which would otherwise occur in the presence of wild-type IGFBP-2. As a consequence, no activation of angiogenic processes were observed in NLSmutIGFBP-2 expressing SHEP cells when implanted onto our in vivo quail chorio-allantoic membrane model. Taken together, these data show for the first time that IGFBP-2 possesses a functional NLS sequence and that IGFBP-2 actively translocates into the nucleus by a classical nuclear import mechanism, involving formation of IGFBP-2 complexes with importin-α. Nuclear IGFBP-2 is required for the activation of VEGF expression and consequent angiogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / genetics*
  • Amino Acid Sequence
  • Cell Line, Tumor
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism
  • Humans
  • Insulin-Like Growth Factor Binding Protein 2 / genetics
  • Insulin-Like Growth Factor Binding Protein 2 / metabolism*
  • MCF-7 Cells
  • Neoplasms / metabolism*
  • Neovascularization, Pathologic / metabolism
  • Nuclear Localization Signals / genetics*
  • Promoter Regions, Genetic
  • Sequence Alignment
  • Vascular Endothelial Growth Factor A / biosynthesis*
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism
  • alpha Karyopherins / metabolism*
  • beta Karyopherins / metabolism

Substances

  • DNA-Binding Proteins
  • IGFBP2 protein, human
  • Insulin-Like Growth Factor Binding Protein 2
  • Nuclear Localization Signals
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • alpha Karyopherins
  • beta Karyopherins
  • DNA