IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells

J Cancer Res Clin Oncol. 2012 Jun;138(6):1045-55. doi: 10.1007/s00432-012-1153-y. Epub 2012 Mar 6.


Objectives: Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells.

Methods: DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1.

Results: Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1 were partially reversed by p21 knockdown in MCF-7 cells. Knockdown of p53 in MCF-7 cells did not influence the growth inhibition, cellular senescence, and p21 expression of the cells in response to IGFBP-rP1 transfection.

Conclusions: Results from this study suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 enhanced expression, which regulated through the p53-independent pathway. IGFBP-rP1 might be one of the key molecules that trigger cellular senescence in breast cancer. Restoration of IGFBP-rP1 function might have therapeutic significance in breast cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology*
  • Cell Cycle Checkpoints / physiology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Growth Processes / physiology
  • Cell Line, Tumor
  • Cellular Senescence / physiology
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • Female
  • G1 Phase / physiology
  • Gene Expression Regulation, Neoplastic
  • Gene Knockdown Techniques / methods
  • Genes, Tumor Suppressor
  • Humans
  • Insulin-Like Growth Factor Binding Proteins / genetics
  • Insulin-Like Growth Factor Binding Proteins / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Resting Phase, Cell Cycle
  • Retinoblastoma Protein / genetics
  • Retinoblastoma Protein / metabolism
  • Signal Transduction
  • Transfection / methods
  • Tumor Suppressor Protein p53 / biosynthesis*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism


  • CDKN1A protein, human
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p21
  • Insulin-Like Growth Factor Binding Proteins
  • Recombinant Fusion Proteins
  • Retinoblastoma Protein
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • insulin-like growth factor binding protein-related protein 1