Radiation-Induced S-Nitrosylation of ER-α Predisposes MCF-7 Breast Cancer Cells to Aggressive Invasion and Migration.

Abstract

Abstract The objective of this study is to decipher the potentials of breast cancer cells that survive radiation exposure at clinical doses to acquire invasive and metastatic determinants. In this study we exposed estrogen receptor positive (MCF-7) and negative (MDAMB-231) breast cancer cells to clinical doses of low LET radiation including doses used as single fraction (2Gy), cumulative dose (10Gy) and scattered dose (10cGy). The cells were exposed to 137Cs source at a dose rate of 1.27 Gy/min. The mock irradiated cells were used as controls. The mRNA transcript level, protein expression and protein-protein interaction were analyzed by QPCR/RT-PCR, immunoblotting, immunoprecipitation and mammalian two-hybrid system, respectively. Intracellular nitric oxide (NO) levels were determined by immunofluorescence and electron spin resonance spectroscopy (ESR) using DAF-FM and Fe-MGD as NO traps, respectively. NO-dependent ER-α s-nitrosylation was determined through immunoprecipitating s-nitrosylated proteins from the cell lysates with an s-nitrosylated-cysteine IgG antibody followed by ER-α western blot analysis. Transactivation of ER-α was measured by EMSA and luciferase reporter assay. Cell invasion and migration were examined by co-culture system using thin inserts.Radiation induces eNOS expression and activation through the phosphorylation of eNOS at Ser1177 site resulting in increased bioavailability of NO in a dose- and time-dependent manner. The three-fold bioavailability of NO s-nitrosylates ER-α leading to a two-fold binding of ER-α to ERE. Paradoxically, this binding did not translate into the transcriptional activation of ER-α dependent gene regulation. Altered structure due to s-nitrosylation of ER-α contributes to enhanced cell invasion and cell migration. In consistent with these results, there was an associated 2.3-fold increase in the expression of MMP-2 and MMP-9 and corresponding TIMP-1 decreased expression leading to a 3.5-fold increase in MMP activity. Radiation, while controlling tumor growth, could simultaneously play a significant role in breast cancer relapse and metastasis through the activation of eNOS and the generation of NO. The bioavailability of NO s-nitrosylates ER-α leading to re-defining the genomic functions of ER-α and thereby impart metastatic transformation potentials to ER-α positive breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6162.