Blocking IL4- and IL13-mediated phosphorylation of STAT6 (Tyr641) decreases M2 polarization of macrophages and protects against macrophage-mediated radioresistance of inflammatory breast cancer

Publication date: Available online 7 December 2017
Source:International Journal of Radiation Oncology*Biology*Physics
Author(s): Omar M. Rahal, Adam R. Wolfe, Pijus K. Mandal, Richard Larson, Sanda Tin, Cristina Jimenez, Dadong Zhang, Janet Horton, James M. Reuben, John S. McMurray, Wendy A. Woodward
PurposeThe purpose of this study was to determine the role of macrophage polarization on the response of IBC cells to radiation and whether modulation of macrophage plasticity can alter radiation response.Methods and MaterialsThe human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "anti-tumor" (M1) or a "pro-tumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 h, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by qPCR.ResultsExpression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, while co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of PRKCZ in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable shRNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture.ConclusionsThese data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by downregulating PRKCZ.

Teaser

Here we show that M2-polarized macrophages upon co-culture with IBC cells, promote radioresistance of IBC cells, and this effect was inhibited by PM37, a phosho-STAT6 inhibitor. M2-macrophages mediated radioresistance of KPL4 IBC cells is associated with increased protein expression of PRKCZ kinase in KPL4 cells, after M2-macrophage co-culture, and this was prevented by PM37-mediated inhibition of M2 polarization of THP1 macrophages prior to coculture with KPL4 cells.


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