PTEN inhibits macrophage polarization from M1 to M2 through CCL2 and VEGF-A reduction and NHERF-1 synergism

Cancer Biol Ther. 2015;16(2):297-306. doi: 10.1080/15384047.2014.1002353.


PTEN has been studied in several tumor models as a tumor suppressor. In this study, we explored the role of PTEN in the inhibition state of polarized M2 subtype of macrophage in tumor microenvironment (TME) and the underlying mechanisms. To elucidate the potential effect in TME, RAW 264.7 macrophages and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. After PTEN was down-regulated with shRNA, the expression of CCL2 and VEGF-A, which are definited to promote the formation of M2 macrophages, have a dramatically increase on the level of both gene and protein in co-cultured RAW 264.7 macrophages. And at the same time, NHERF-1 (Na(+)/H(+) exchanger regulating factor-1), another tumor suppressor has a similar tendency to PTEN. Q-PCR and WB results suggested that PTEN and NHERF-1 were consistent with one another no matter at mRNA or protein level when exposed to the same stimulus. Coimmunoprecipitation and immunofluorescence techniques confirmed that PTEN and NHERF-1 were coprecipitated, and NHERF-1 protein expression was properly reduced with rCCL2 effect. In addition, cell immunofluorescence images revealed a profound transferance, in co-cultured RAW 264.7 macrophages, an up-regulation of NHERF-1 could promote the PTEN marked expression on the cell membrane, and this form for the interaction was not negligible. These observations illustrate PTEN with a certain synergy of NHERF-1, as well as down-regulation of CCL2 suppressing M2 macrophage transformation pathway. The results suggest that the activation of PTEN and NHERF-1 may impede the evolution of macrophages beyond the M1 into M2 phenotype in tumor microenvironment.

Keywords: CAFs, cancer associated fibroblasts; CCL2; CM, complete medium; CXCL, the chemokine (C-X-C motif) ligand; CXCR3, Chemokine (C-X-C motif) receptor 3; FAK, focal adhesion kinase; NHERF-1; NHERF-1, Na+/H+ exchanger regulating factor1; PTEN; PTEN, phosphatase and tensin homolog deletedon chromosome 10; SCC, squamous cell carcinoma; TAM; TAMs, tumor-associated macrophages; TSN, tumor culture supernatant; co-culture; transformation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Membrane / metabolism
  • Cell Movement / genetics
  • Cell Proliferation
  • Chemokine CCL2 / metabolism*
  • Down-Regulation
  • Gene Expression Regulation, Neoplastic
  • Gene Knockdown Techniques
  • Humans
  • Immunohistochemistry
  • Macrophages / metabolism*
  • Mice
  • Neoplasms / genetics
  • Neoplasms / metabolism
  • PTEN Phosphohydrolase / genetics
  • PTEN Phosphohydrolase / metabolism*
  • Phosphoproteins / metabolism*
  • Sodium-Hydrogen Exchangers / metabolism*
  • Tumor Microenvironment / genetics
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism*


  • Chemokine CCL2
  • Phosphoproteins
  • Sodium-Hydrogen Exchangers
  • Vascular Endothelial Growth Factor A
  • sodium-hydrogen exchanger regulatory factor
  • PTEN Phosphohydrolase