Modulation of Glucocorticoid Induced Tumor Necrosis Factor Receptor (GITR)-GITR Ligand (GITRL) Interaction in Breast Cancer Cells Under the Control of Ataxia Telangiectasia Mutated (ATM) Promoter

Abstract

Treatment response for basal-like breast cancers (BLBC) is limited and this aggressive breast cancer sub-type has poor prognosis and high mortality. Our first aim is to investigate ATM activity in BLBC cell lines with ionizing radiation. Our second aim is to study the viability of BLBC cells by the GITR-GITRL interaction while examining the expression levels of GITR and GITRL with radiation. ATM expression levels in basal-like (MDA-MB-231, HCC38, MDA-MB-468) and luminal (MCF-7, BT-474, SK-BR-3) breast cancer cell lines were analyzed with RT-PCR and found similar. Increase in ATM (S1981) phosphorylation in BLBC cells with ionizing radiation has been demonstrated with Western Blot experiments. HCC38 cells transfected with “pATM-GL3” Luciferase reporter plasmid showed high basal and post-radiation ATM activity. Although there is no difference in ATM mRNA levels, changes in protein levels has been observed, suggesting a post-transcriptional control mechanism. GITR and GITRL expressions in BLBC cells were investigated via RT-PCR and there was no change in expression levels with radiation. While MDA-MB-231 and MDA-MB-468 cell lines show high GITRL expression, HCC38 cell line was GITR positive, with both RT-PCR and flow cytometry. GITR+ HCC38 cells were incubated with recombinant GITRL protein at different serum concentrations (1% and 10%) and the change of cancer cells’ viability, proliferation and amount of metabolically active viable cells were investigated with DRAQ7 staining, CFSE assay and MTT assay, respectively. Even though GITR stimulation only has not changed viability and proliferation of HCC38 cells, both ionizing radiation and GITR stimulation had a cumulative effect on cell viability. When cell death was assayed with DRAQ7 staining, a decrease in viability of the cells was observed, which were simultaneously exposed to both 80 ng/ml rGITRL and 5 Gy ionizing radiation. As a result, this study demonstrated that cumulative effect of GITR stimulation and ionizing radiation may affect the viability of breast cancer cells.